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Tubulin-based fusion proteins as multifunctional tools
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Tubulin-based fusion proteins as multifunctional tools
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1
TUBULIN-BASED FUSION PROTEINS AS MULTIFUNCTIONAL TOOLS
by
Jordan Despanie
A Dissertation presented to the
Department of Pharmacology and Pharmaceutical Sciences
University of Southern California
Faculty of the USC Graduate School
In partial fulfillment of the
Requirements for the degree
DOCTOR OF PHILOSOPHY
PHARMACEUTICAL SCIENCES
May 2019
Copyright 2019 Jordan Despanie
2
Dedication
I would like to thank my mentor, Dr. J. Andrew MacKay, for the unstinting support and
tireless effort he has expended towards helping me realize my dream of obtaining a
doctorate despite my medical adversities. I must also extend my gratitude to the
committee members ― Dr. Sarah Hamm-Alvarez, Dr. Curtis Okamoto, and Dr. Darcy
Spicer ― for their understanding and crucial feedback alongside the time devoted to
reviewing this thesis. I am likewise grateful to all past and current members of the
MacKay Lab as well as fellow collaborators ― namely, Dr. Nicos Petasis, Dr. Nikita
Vlasenko, Robert Nshimiyimana, and Farhan Chowdhury for lending their synthesis
expertise in generating the colchicine-based analogues ― who have aided me during
this scientific journey. I would finally like to thank my family and friends, most especially
the Guss family, who have together served as mountains of stability throughout the
ever-shifting odyssey that is life.
3
Table of Contents
Dedication ...................................................................................................................... 2
List of Tables ................................................................................................................. 7
List of Figures................................................................................................................ 8
Abstract ........................................................................................................................ 12
Chapter 1: Introduction ............................................................................................... 13
1.1 Nanomedicine .................................................................................................. 13
1.2 Tubulins and the microtubule network .............................................................. 17
1.3 Elastin-like polypeptides ................................................................................... 23
1.3.1 Molecular cloning ..................................................................................... 27
1.3.2 Protein expression and physicochemical characterization ....................... 29
1.4 Applications of elastin-like polypeptides ........................................................... 36
1.4.1 Cancer ..................................................................................................... 37
1.4.2 Ocular diseases ....................................................................................... 46
1.4.3 Cardiovascular diseases .......................................................................... 50
1.4.4 Maternal-fetal medicine............................................................................ 52
1.4.5 Diabetes ................................................................................................... 53
1.4.6 Regenerative medicine ............................................................................ 58
4
1.4.7 Infectious diseases .................................................................................. 59
1.4.8 Gene delivery ........................................................................................... 63
1.4.9 Respiratory diseases ............................................................................... 65
1.5 ELPs as colchicine nanocarriers ...................................................................... 66
1.6 ELPs as purification tools ................................................................................. 69
1.7 ELPs as intracellular switches .......................................................................... 70
Chapter 2: Materials and Methods ............................................................................. 73
2.1 Materials .......................................................................................................... 73
2.2 Methods ........................................................................................................... 73
2.2.1 Gene design and synthesis of bacterially expressed tubulin-ELPs .......... 73
2.2.2 Western blot analysis of tubulin-ELP inclusion bodies ............................. 74
2.2.3 Expression and purification of soluble tubulin-ELPs ................................ 75
2.2.4 Determination of protein purities and transition temperatures .................. 77
2.2.5 Secondary structure determination using circular dichroism .................... 78
2.2.6 Light scattering analyses of nanoparticles ............................................... 79
2.2.7 MALDI-ToF analysis of molecular weights ............................................... 80
2.2.8 Drug retention via extended dialysis ........................................................ 80
2.2.9 Tubulin polymerization ............................................................................. 81
2.2.10 Negative transmission electron microscopy of nanoparticles ................... 81
5
2.2.11 Pronase treatment of tubulin-ELPs .......................................................... 82
2.2.12 Gene design of mammalian expressed tubulin-ELP ................................ 82
2.2.13 Transfection and visualization of a mammalian tubulin-ELP .................... 83
Chapter 3: Results....................................................................................................... 84
3.1 Bacterially expressed tubulin-ELPs can be obtained in the soluble fraction ..... 86
3.2 Tubulin fusions gain ELP-based thermoresponsive properties ........................ 91
3.3 Tubulin fusions retain secondary structures resembling a bovine
tubulin control ................................................................................................. 96
3.4 Soluble tubulin fusions derived from a hydrophilic ELP are stable
across a broad temperature range ................................................................. 101
3.5 Tubulin fusions are not amenable to MALDI-ToF analysis ............................. 104
3.6 Tubulin-ELPs assemble into nanoparticles rich in tubulin
moieties relative to a bovine tubulin control.................................................... 107
3.7 Tubulin fusions exhibit colchicine-induced quenching .................................... 116
3.8 A soluble tubulin fusion does not enhance colchicine retention in vitro .......... 118
3.9 αβ tubulin fusions recapitulate polymerization kinetics yet remain
unresponsive to microtubule inhibitor ligands................................................. 119
3.10 Tubulin fusion nanoparticles predominantly appear as oligomers
via negative TEM imaging ............................................................................. 124
6
3.11 Microtubules derived from αβ-tubulin fusions are not apparently
visualized via negative TEM under polymerizing conditions ....................... 131
3.12 A kinetics-based proteolytic treatment cleaves TβS96 more
effectively than TαS96 or TαβS96 ................................................................. 136
3.13 A mammalian-expressed tubulin fusion integrates into the microtubule
network ......................................................................................................... 139
Chapter 4: Discussion and Future Directions......................................................... 140
4.1 ELPs as colchicine nanocarriers .................................................................... 140
4.2 ELPs as purification tools ............................................................................... 147
4.3 ELPs as intracellular switches ........................................................................ 152
4.3.1 Future Directions ................................................................................... 155
4.4 Conclusions.................................................................................................... 157
Acknowledgements ................................................................................................... 164
List of Abbreviations ................................................................................................. 164
References ................................................................................................................. 168
7
List of Tables
Table 1: Elastin-like polypeptide nanomedicines under commercial investigation ........ 56
Table 2: Physical properties of ELP protein polymers with and without tubulins .......... 84
Table 3: Proteins evaluated via SDS-PAGE in Figure 12 ............................................. 88
Table 4: Proteins evaluated via SDS-PAGE in Figure 13 ............................................. 90
Table 5: Quantification of temperature-concentration phase diagram in Figure 13C .... 93
Table 6: Quantification of temperature-concentration phase diagram in Figure 14B .... 95
Table 7: BeStSel estimated secondary structure content ........................................... 100
Table 8: Quantification of SEC-MALS Chromatogram in Figure 22 ............................ 109
Table 9: Quantification of SEC-MALS chromatogram in Figure 23A .......................... 111
Table 10: Quantification of SEC-MALS chromatogram in Figure 23B ........................ 113
Table 11: Quantification of SEC-MALS chromatogram in Figure 24 ........................... 115
Table 12: Quantification of Negative TEM Data from Figures 31 to 33 ....................... 129
Table 13: Quantification of Negative TEM Data from Figures 35 to 37 ....................... 135
Table 14: Summary of Experimental Approaches and Results ................................... 158
8
List of Figures
Figure 1: Tubulin polymerization and the binding sites of microtubule inhibitors .......... 17
Figure 2: Molecular structures of the microtubule inhibitors investigated
in this dissertation. ......................................................................................... 21
Figure 3: Depiction of reversible phase separation by elastin-like polypeptides ........... 25
Figure 4: Uptake and degradation of ELP nanoparticles .............................................. 34
Figure 5: Various applications of ELPs as nanomedicines ........................................... 36
Figure 6: Design of elastin-like polypeptide nanoparticles that carry an anti-proliferative
drug at both their core and corona ................................................................ 43
Figure 7: Lacritin-ELP nanoparticles heal corneal wounds in mice .............................. 47
Figure 8: NIR tomography of (GLP-1)-ELP after subcutaneous injection ..................... 55
Figure 9: Serum half-life of
Nt
TNF-VHHELP and neutralization of LPS/D-Gal toxicity
by
Nt
TNF-VHHELP ........................................................................................... 60
Figure 10: Overview of α and β tubulins genetically modified with ELPs as
potentially polymerization competent proteins ............................................. 70
Figure 11: Tubulin-ELPs are sequestered into inclusion bodies when expressed in
BLR E. coli cells .......................................................................................... 86
Figure 12: TβSI and TβS96 exhibit soluble expression and can be obtained at
high purity levels ......................................................................................... 87
9
Figure 13: Differential staining reveals soluble expression of TαS96 and TβS96
at high yields .............................................................................................. 89
Figure 14: TβSI and TβS96 gain ELP-mediated thermoresponsive behaviors ............. 91
Figure 15: TαS96 and TβS96 remain stable at physiologically relevant temperatures
similar to S96 .............................................................................................. 94
Figure 16: Tubulin fusions retain secondary structures resembling a bovine tubulin
control......................................................................................................... 96
Figure 17: Deconvolution confirms that soluble tubulin fusions exhibit secondary
structures similar to bovine tubulin .............................................................. 99
Figure 18: TβS96 is a more physiologically relevant nanoformulation than TβSI ....... 101
Figure 19: TαS96 exhibits hydrodynamic radii slightly larger than TβS96 .................. 102
Figure 20: MALDI-ToF analysis confirms the molecular weight of S96
but not TβS96 ............................................................................................ 104
Figure 21: MALDI-ToF analysis cannot accurately confirm the molecular weight
of TβSI ....................................................................................................... 105
Figure 22: Bovine tubulin exhibits the morphological features of a
monomer in PBS ....................................................................................... 107
Figure 23: TβSI possesses three distinct fractions with disparate morphological
features ..................................................................................................... 110
Figure 24: TαS96 and TβS96 consist of tubulin-compacted oligomers ...................... 114
10
Figure 25: Colchicine absorbs tubulin fluorescence emission in a concentration-
dependent manner .................................................................................... 116
Figure 26: TβS96 does not enhance colchicine retention........................................... 117
Figure 27: TαS96 and TβS96 appear to mimic microtubule dynamics while also
potentially demonstrating patterns suggestive of self-polymerization ....... 119
Figure 28: Paclitaxel does not seemingly enhance the polymerization
kinetics of TαβS96 ..................................................................................... 121
Figure 29: Colchicine and three semisynthetic analogues appear incapable of altering
TαβS96 polymerization kinetics ................................................................. 122
Figure 30: Colchicine and three semisynthetic analogues induce destabilization
followed by polymerization recovery in the presence of bovine tubulin .... 123
Figure 31: Negative TEM reveals that TαS96 exists predominantly as high aspect ratio
nanoparticles ............................................................................................. 124
Figure 32: Negative TEM reveals that TβS96 exists predominantly as high aspect ratio
nanoparticles ............................................................................................. 125
Figure 33: Negative TEM demonstrates that Bovine Tubulin exists as spherical
particles ..................................................................................................... 126
Figure 34: Negative TEM micrograph of a uranyl acetate-stained blank grid ............. 127
Figure 35: Negative TEM micrograph reveal that unpolymerized bovine tubulin remain
the predominant population even with paclitaxel present .......................... 131
11
Figure 36: Bovine tubulin forms paclitaxel-stabilized microtubules under polymerization
conditions .................................................................................................. 132
Figure 37: In the presence of paclitaxel, TαβS96 does not apparently
form microtubules ...................................................................................... 133
Figure 38: Pronase cleaves TβS96 more effectively than TαS96 or TαβS96 ............. 137
Figure 39: A mammalian expressed β-tubulin ELP fusion intercalates
into an existing microtubule network ......................................................... 139
12
Abstract
This dissertation investigates the prospect of harnessing tubulins genetically fused with
elastin-like polypeptides as multifunctional tools. Tubulins are dynamic proteins which
assemble into polymers called microtubules to participate in critical roles throughout the
cell such as cytoskeletal stability, intracellular transport, and mitosis. Given their
physiological importance, these native protein polymers have been clinically exploited
as targets for small molecule drugs capable of interfering with the microtubule network.
Elastin-like polypeptides (ELPs), meanwhile, constitute a genetically engineered class
of ‘protein polymers’ derived from human tropoelastin. They exhibit a reversible phase
separation whereby samples remain soluble below a transition temperature (Tt) but form
amorphous coacervates above Tt. ELPs phase behavior has many possible applications
in purification and nanoassembly which can be further expanded by fusing this
thermoresponsive moiety to native proteins. Recombinant synthesis additionally affords
precise control over an ELP fusion protein’s architecture and molecular weight, resulting
in functionalized protein polymers with uniform physicochemical properties suited to the
design of tools relevant to nanomedicine and synthetic biology. The studies featured
within this dissertation therefore explore tubulin-ELP fusion proteins as multifunctional
tools with three broad applications ― namely, as colchicine nanocarriers, tubulin-based
purification tools, and intracellular switches. Although unsuccessful as colchicine
nanocarriers, these ELP-based tools have potentiated a non-chromatographic method
for the purification of recombinantly expressed tubulins and offer the possibility of
thermally regulating microtubule dynamics within an intracellular milieu.
13
Chapter 1
Introduction
1.1 Nanomedicine
While ambitious visions for nanomedicine have outpaced their practical applications,
nanomedicine has had a significant impact on drug delivery systems. First envisioned in
1906 by Paul Ehrlich(Strebhardt and Ullrich, 2008), these novel drug delivery systems
are aimed at improving clinical outcomes through innovations in particle size, shape,
multifunctionality, site-directed delivery, and the reduction of toxicity(Hoffman, 2008).
The therapeutic index, defined as a ratio between toxic and effective doses, is one
measure by which nanomedicines may be optimized. Drug toxicity depends on duration,
concentration, and total exposure to drug. In particular, many chemotherapeutics have
low therapeutic indices caused by dose-limiting side-effects in normal tissues. While
raising the dose of an approved free drug can improve efficacy, off-target effects render
this unsafe. Thus, a major rationale for exploring nanomedicines is that they can
enhance the therapeutic index compared to their free drug counterpart.
One potential advantage of nanomedicine includes the capacity to construct materials
that ferry diverse drugs to sites of disease for controlled release and/or site-directed
delivery. By tuning the size and architecture of nanoparticles, for instance, it may be
possible to enhance drug residence times and augment the therapeutic index over what
could be achieved with free drug alone. Breakthroughs have been most prominent in
the oncology space with the approval of nanoformulations, such as liposomal
14
doxorubicin and albumin-bound paclitaxel, aimed at exploiting the enhanced
permeability and retention (EPR) effect sometimes seen in tumors (Cho et al., 2008a;
Greish, 2007). Alternatively, with over 50% of all anti-cancer drugs doubling as
substrates of the P-glycoprotein efflux pump, nanoparticles are a compelling solution to
overcome multidrug resistance (Sharom, 1997).
Extensive efforts to expand the utility of nanoformulations have relied on grafting
polymers to their surface. Liposomes are among the best-characterized nanomedicine
platforms (Malam et al., 2009). For early liposomal formulations, rapid opsonization and
detection by the mononuclear phagocyte system, which removes foreign bodies from
the blood, hampered the mean residence time and therapeutic efficacy (Owens and
Peppas, 2006). To prevent opsonization, protective polymers were later developed to
sterically shield nanoparticles, which propelled liposomes to clinical approval twenty
years ago (Barenholz, 2012). Based on data from liposomes and other similarly sized
particles, the surface properties for new carriers should be optimized to minimize
opsonization, prevent complement activation, and eliminate clearance by the
mononuclear phagocyte system. All of these factors can be strongly influenced by
composition and architecture of polymers at the nanoparticle surface. Thus, advances in
nanomedicine have been closely tied to the development of polymers for biological
applications.
As nanomaterials, high molecular weight polymers can solubilize hydrophobic drugs
(Duncan, 2003; Mitragotri et al., 2015). Polymer-drug conjugation involves the
15
appending of a water-soluble polymer onto the drug of choice (Vicent and Duncan,
2006). Polymers with large hydrodynamic radii can prevent renal filtration and extend
the drug mean residence time. If these polymers are amphiphilic, they can also directly
mediate nanoparticle assembly (MacKay et al., 2009; Sakai-Kato et al., 2015). As such,
vast arrays of polymeric species, ranging from natural to synthetic, have been studied
as drug carriers. Chemical polymerization produces mixtures of polymers with differing
chain length, which necessitates statistical definitions of their polydispersity. The
polydispersity index (PDI) is defined as the ratio of the mass-average molecular weight
(Mw) to the number-average molecular weight (Mn) and characterizes the heterogeneity
in a system. A monodisperse polymer possesses a PDI approaching 1 while
polydisperse polymers have PDI greater than 1. The PDI is highly dependent on the
mechanism of the polymerization reaction (Rogošić et al., 1996). As polymers of
differing molecular weights have various fates in the body, it is evident that the PDI
should be controlled to the greatest extent possible (Yamaoka et al., 1995). Additional
aspects of polymer nanomaterials to be considered include immunogenicity,
biodegradability, efficiency of encapsulation and drug loading, as well as stability on the
shelf and in the body.
Synthetic polymers, e.g. polyethylene glycol (PEG) and polycaprolactone (PCL), have
seen widespread use as biomaterials owing to their low immunogenicity and high
biocompatibility. Drawbacks related to polydispersity, linkage stability, and limited
carrying capacity(Duncan, 2006; Veronese, 2001) unfortunately limit the delivery
applications of these platforms. More recent advances have since focused on the
16
fabrication of hybrid nanocarrier systems. Block copolymers, for instance, can
spontaneously self-assemble into nanostructures in the form of micelles, electrostatic
complexes, and polymersomes due to their amphiphilic properties(Hoffman, 2008).
Compared to conventional polymers, biomaterials derived from recombinant proteins
may serve as viable alternatives. The molecular techniques underpinning these ‘protein
polymer’ technologies were first detailed in 1986(Ferrari et al., 1993; Srinivasan and
Kumar, 2012). Archetypal peptides include leucine zippers(Hakoshima, 2001), collagen-
like polymers(Luo and Kiick, 2013), extended recombinant polypeptide (XTEN)
polymers(Schellenberger et al., 2009), silk-like polypeptides (SLPs)(Valluzzi et al.,
2002), and elastin-like polypeptides (ELPs)(Urry, 1997). These protein polymers,
comprised of repeating motifs sourced from either natural or de novo engineered amino
acid sequences, are generated via genetic engineering and recombinant biosynthesis.
This offers an exquisite level of precision and tunability with regards to the length,
molecular weight, sequence, and monodispersity of the resulting material. Owing to
their origin as natural polypeptide chains, protein polymers can be biocompatible and
biodegradable, leaving only small peptides and amino acids as their metabolic
byproducts(Frandsen and Ghandehari, 2012).
Genetic encoding, meanwhile, grants structural and functional control over molecular
features such as secondary structures, targeting motifs, and drug conjugation sites. This
provides a compelling rationale for the systematic construction of libraries varying by
specific amino acid residues. The addition of natural peptides and protein domains to
17
these polymers creates fusion proteins which often maintain the function and activity of
the parent macromolecule(Floss et al.,
2010b). Finally, the low costs of large-scale
production in biological systems render
recombinant protein polymers amenable to
process scale-up(Hassouneh et al., 2012).
1.2 Tubulins and the Microtubule
Network
Microtubules play an integral role in the
eukaryotic cell by providing cytoskeletal
stability while also participating in activities
as varied as motility, intracellular transport,
and cell division(Dumontet and Jordan,
2010; Jordan and Wilson, 2004). These
complex biopolymers are comprised of αβ-
tubulins which exhibit intricate polymerization
dynamics between soluble heterodimers and
polymerizing microtubules essential to their
cellular activities(Akhmanova and Steinmetz,
2015) (Figure 1A). During mitosis,
microtubule dynamics prove crucial to the
formation of the mitotic spindle apparatus, an organelle responsible for the segregation
Figure 1. Tubulin polymerization
and the binding sites of
microtubule inhibitors. (A) Soluble
tubulin dimers consisting of α and β
subunits polymerize to form
microtubules. β-tubulin subunits serve
as the + end for further microtubule
growth. (B) Vinca alkaloids, such as
vinblastine, bind to the + end of
growing microtubules and induce
depolymerization. Colchicine acts on
soluble tubulin dimers to prevent
polymerization while taxanes like
paclitaxel bind to the inner lumen of
microtubules. Image adapted from
Dumontet and Jordan, 2010.
18
of aligned chromosomes prior to cell division. This polymerization is mediated through
the reversible incorporation of tubulin dimers at a region on microtubule ends known as
a GTP cap. More specifically, hydrolysis of β-tubulin bound GTP into the tubulin–GDP–
inorganic phosphate complex occurs shortly after addition, thereby conferring a
stabilizing effect termed ‘microtubule rescue’ on the available end and permitting further
self-assembly. The GTP cap, however, eventually dissociates from the microtubule,
leaving a protofilament comprised of GDP-tubulin intrinsically prone to disassembly
during ‘microtubule catastrophe’(Gardner et al., 2013). Both the plus end with β-tubulin
exposed and the minus end containing α-tubulin possess GTP caps capable of
switching between phases of growth and shortening, although they exhibit differing
microtubule dynamics. The plus end undergoes this process more rapidly and
depolymerizes in the absence of the GTP cap whereas the opposite holds true of its
more stable minus end counterpart. Overall, this cyclical model of rescue and
catastrophe — collectively defined as dynamic instability — proves critical to a broad
range of biological processes including maintenance of cellular integrity, vesicular
transport, cell signaling, and mitosis(Janmey, 1998; Topham and Taylor, 2013). Despite
advances across the realm of molecular and cell biology, however, the mechanisms
governing dynamic instability remain poorly understood.
The functional importance of tubulins throughout evolutionary history has prompted the
emergence of various tubulin isotypes containing related yet distinct amino acid
sequences(Berrieman et al., 2004; Huzil et al., 2006). In humans, for instance, this
heterogeneity manifests as 8 isotypes of α-tubulin and 7 isotypes of β-tubulin, each
19
expressed at varying levels across a broad selection of cells and tissues(Parker et al.,
2017). βI-tubulin, the first isotype explored in this project, is encoded by the TUBB gene
at the 6p25 locus and exhibits constitutive expression, rendering it the most commonly
expressed isotype in both normal and cancerous cells(Wang et al., 2005). This isotype
also typifies the protein domains found in all other variants with the N-terminal and
intermediate domains comprising a globular core; the C-terminal domain, meanwhile,
behaves as an intrinsically disordered tail possessing a net negative charge due to its
highly-conserved abundance of glutamate residues(Janke, 2014). Interestingly, cancer
cells often display the ability to sequester native tubulins in favor of upregulating
isotypes unrelated to the cell lineages from which they are derived in a process known
as isotype switching (alternatively called tubulin autoregulation)(Gay et al., 1989). βIII-
tubulin, an isotype confined to cells of neuronal origin, particularly correlates with
enhanced resistance to MTIs across various breast cancers including TNBC(Hari et al.,
2003; Lebok et al., 2016). The second tubulin protein investigated in order to study
microtubule polymerization during this project was α1a-tubulin; encoded by the TUBA1A
gene at the 12q13.12 locus, α1a-tubulin is predominantly expressed within the
brain(Aiken et al., 2017; Belvindrah et al., 2017).
Given the array of roles played by microtubules in the cell, several molecular events
(e.g. temperature range, tubulin concentrations, GTP supply) underpin microtubule
assembly and disassembly including those which have yet to be elucidated(Prasad et
al., 1992). As an example, in vitro studies have established that microtubules growing
for longer periods tend to undergo catastrophe at greater rates relative to their younger
20
counterparts; catastrophe may also be triggered by the action of forces, such as the cell
cortex, competing against growing microtubule tips. The mechanisms behind rescue
seem to be even more nebulous since its occurrence in vitro is not necessarily related
to tubulin concentrations. It has therefore been suggested that rescue might be
promoted by local features like GTP-tubulin ‘islands’ which mimic the stabilizing GTP
cap in the microtubule lattice to halt disassembly. The composition of tubulin isotypes
likewise contributes to microtubule assembly kinetics. Even small differences in the
binding energies, electrostatic potentials, or chemical affinities of these particular
isotypes can have an outsized effect on variations in microtubule rescue rates and
catastrophe frequencies. It has nevertheless been demonstrated that tubulins freely
polymerize into heterogeneous microtubules independent of their isotype
composition(Lewis et al., 1987). Prior investigations have further revealed that tubulin
coassembly can even occur in an interspecies context including hybrid carrot/pig
microtubules in vitro and mouse microtubules that incorporate chimeric chicken/yeast
21
tubulins following transfection(Bond et al., 1986; Slabas et al., 1980).
Since disruption of microtubule dynamics can induce mitotic arrest and initiate cell
death, small molecule ligands targeting tubulin have been successfully employed as
potent pharmacological agents and form the basis of multiple chemotherapy regimens
(Figure 1B). The molecular structures of the small molecule drugs investigated in this
dissertation are depicted (Figure 2)(Dong et al., 2016). Microtubule inhibitors primarily
fall into two categories: i) compounds irreversibly stabilizing microtubule assembly (e.g.
taxanes, epothilones); and ii) agents that destabilize and ultimately depolymerize
microtubules (e.g. colchicinoids, vinca alkaloids)(Dumontet and Jordan, 2010; Jordan
and Wilson, 2004). Although some of these small molecules have been employed as
Figure 2. Molecular structures of the microtubule inhibitors investigated in this
dissertation. (A) Paclitaxel (MW=853.91 Da); (B) Colchicine (MW=399.44 Da); (C)
CBA (MW=500.59 Da); (D) CDA (MW=464.56 Da); (E) CMA (MW=511.62 Da).
MW refers to molecular weight in daltons.
22
chemotherapeutics, they possess several clinical drawbacks including drug resistance,
poor water solubility, and pronounced off-target toxicities(Rowinsky and Donehower,
1991). A demand therefore exists for novel cytotoxic drugs with enhanced safety
profiles alongside innovative delivery strategies to ferry these potent agents more
effectively(Cho et al., 2008a; Wang et al., 2013).
Tubulin’s therapeutic relevance has similarly driven extensive biomedical investigations,
although isolation and purification of research-grade material from surrogate
mammalian sources (e.g. bovine, porcine) invariably remains a laborious, cost-intensive
undertaking(Gell et al., 2011). Reports detailing the recombinant expression of human
tubulins from E. coli, however, effectively illustrate that synthetic biology can solve the
issue of generating isotypically pure tubulins from any species(Friesen et al., 2012;
Mane et al., 2013; Minoura et al., 2013). Nevertheless, bacterially-produced tubulins are
prone to being sequestered into inclusion bodies thus necessitating cumbersome
solubilization and refolding steps prior to yielding purified material(Singh and Panda,
2005; Yang et al., 2011). A more facile means of obtaining soluble, active recombinant
tubulin proteins would therefore serve as a boon for researchers conducting
biochemical and biophysical studies in this space.
23
1.3 Elastin-like Polypeptides
Elastin is a polymeric extracellular matrix (ECM) protein, found in tissues as varied as
the skin, lungs, blood vessels, and cartilage, which underpins the protractible nature of
vertebrate tissues(Baldock et al., 2011; Partridge et al., 1955; Urry et al., 1995). Though
only one gene encodes the ~60 kDa soluble precursor, tropoelastin, it exists as
polymorphs containing repetitive hydrophobic motifs, largely valine and alanine,
denoting elastomeric domains. Other amino acids present at significant levels include
glycine and proline, which disrupts alpha helix and beta-sheet formation. These
elastomeric domains occur between distinct peptides that are involved in crosslinking
other tropoelastin monomers through the action of lysyl oxidase on lysine residues
(Ciofani et al., 2014b). As biosynthesis and extrusion into the ECM proceed, the final
products generated are insoluble elastin fibrils. Pioneering studies first revealed the
temperature-sensitive nature of hydrolyzed α-elastin(Urry et al., 1969). The protein
remained soluble below 25°C, but when heated to 37°C, elastin phase-separated into a
secondary amorphous phase known as a coacervate. Interestingly, the investigators
further noted that this process was completely reversible. Those findings eventually
facilitated the first chemical synthesis of an elastin-like polypeptide prior to the
emergence of molecular biology as a discipline(Cox et al., 1974).
Elastin-like polypeptides (ELPs) are an artificial, biomimetic class of protein polymers
inspired by the recurring hydrophobic motifs of tropoelastin(Meyer and Chilkoti, 1999).
Due to their broad range of applications, including in drug delivery and tissue
24
engineering, ELPs have attracted attention throughout the scientific community(Chilkoti
et al., 2002; Mackay and Chilkoti, 2008; Meyer and Chilkoti, 2002). The canonical ELP
unit consists of a hydrophobic, five amino acid motif (Val-Pro-Gly-Xaa-Gly)n where the
guest residue, Xaa, specifies any amino acid and n determines the number of
pentapeptide repeats. Proline is usually avoided at the fourth residue since its presence
can interfere with coacervation. It should be noted, however, that inclusion of amino
acid side chains capable of enhancing functionality does not necessarily interfere with
ELP phase behavior. The addition of tyrosine to facilitate spectrophotometric
analysis(Pastuszka et al., 2012) or lysine for crosslinking(Lim et al., 2007; Reichheld et
al., 2014) are two examples. Furthermore, ELPs with other repeat motifs beyond the
one described have similar properties. Examples may range from other pentapeptides
(e.g. IPGVG) to heptapeptide (e.g. LGAGGAG) and nonapeptide (e.g. LGAGGAGVL)
repeat sequences(Kowalczyk et al., 2014). A primary aspect of ELP biomaterials
involves their ability to reversibly form coacervates following temperature changes
(Figure 3A). This feature is known as the critical transition temperature (Tt) and can be
explained thermodynamically in terms of the Gibbs free energy (ΔG = ΔH – TΔS). If ΔG
during a temperature transition (ΔGt) is zero, then ΔHt = TtΔSt which can be rearranged
to Tt = ΔHt/ΔSt. The increase in order of ELPs at Tt might appear to contradict the
second law of thermodynamics — namely that the order of a system has an inverse
relationship with temperature — but the complete system consisting of protein polymer
and water must be considered. In the absence of a stimulus, ELPs remain soluble in
aqueous solutions as random coils and have been described as intrinsically disordered
proteins(Roberts et al., 2015). The hydrophobic side chains of VPGXG remain
25
surrounded by ordered water molecules existing in a low entropy state. Once the
temperature rises above Tt, water molecules clustered around the hydrophobic amino
acids are expelled into the bulk
water phase. This favors a gain in
solvent entropy and allows non-
polar side chains to form intra- and
intermolecular interactions with
neighboring ELP molecules.
Hydrophobic interactions,
meanwhile, facilitate folding and
dynamic assembly into more
ordered secondary structures as
type II β-turn spirals (Urry, 1997).
Employing the model protein
sequence (GVGVP)n, Urry
demonstrated that these
hydrophobic associations are
responsible for a shift from
complete solubility in pure water at
20°C to a state of phase separation at 40°C with a dynamic structured state of 63%
water/37% polypepeptide by weight at physiological temperatures. The self-assembled
form is subsequently denatured at 70°C to a disordered state consisting of 32%
Figure 3. Depiction of reversible phase
separation by Elastin-like polypeptides
(ELPs). (A) ELPs are soluble below a
transition temperature, Tt, but undergo
coacervation at temperatures above Tt. (B) The
linear relationship between Tt and
concentration can be studied by measuring
optical density as a function of temperature.
Three different ELPs of varying length and
hydrophobicity phase separate above the
indicated lines.
26
water/68% polypeptide (Urry et al., 1985). This phase separation phenomena produces
a distinct relationship between the hydrophobicity of an ELP and its transition
temperature. In fact, Urry maintains that every interaction and/or modification of which a
polymer is capable can be characterized as a function of its effect on Tt (Urry, 1997).
The higher the molecular weight or hydrophobicity of an ELP, such as the amino acid-
based hydrophobicity scale, the lower the energy required for surrounding water
molecules to enter the bulk water and thus the lower the heat required to induce
hydrophobic assembly(Urry et al., 2010). In short, ELPs with high molecular weights
and/or hydrophobic guest residues exhibit lower transition temperatures than ELPs with
low molecular weights and/or hydrophilic guest residues. (Figure 3B).
This phase separation property is completely reversible like an on/off switch at
temperatures below the Tt, making ELPs an attractive scaffold as a smart biomaterial
that can respond to temperature(Pastuszka et al., 2014). ELPs can be clinically
designed to exploit their thermal responsiveness. One example is an ELP that remains
soluble at room temperature for ease of injection but phase separates at 37°C within the
target tissue to form an insoluble depot(McDaniel et al., 2010a). The sharp (2-3°C
range) phase separation observed can be tuned to occur between 0-100°C and exists
primarily as a function of guest residue hydrophobicity, molecular weight, and
concentration(Valiaev et al., 2008).
One study suggests that the presence of mild detergents in solution does not curtail
phase separation(Thapa et al., 2013). Phase separation for certain ELPs may also be
27
triggered by external factors such as alterations in ionic strength. Increasing
concentrations of a kosmotropic salt, for instance, reduces the ELP Tt(Cho et al.,
2008b). Modeling approaches that involve the prediction of transition temperatures
further allow investigators to tune ELP design(Christensen et al., 2013). In addition,
certain ELP derivatives have also been described that can induce phase separation in
response to pH changes resulting from ionizable guest residues(Urry, 1993), electrical
current(Jung et al., 2006), redox triggers(Urry et al., 1992), magnetism(Ciofani et al.,
2014a), and light(Shimoboji et al., 2002).
1.3.1. Molecular Cloning
Concatemerization was the original technique employed to genetically engineer libraries
of ELP(Haider et al., 2005). This method relies on the self-ligation of repetitive genetic
sequences thus creating oligomers of various lengths through single-step synthesis.
Although offering a rapid means of ELP gene construction, the primary drawback
remains the fact that absolute control over length is forfeited. In other words, genes
constructed through concatemerization exist as a distribution of DNA oligomers with
varying chain lengths, rather than allowing a distinct length to be specified. While each
bacterial colony arises from a single length of ELP gene, in practice it is difficult to target
a gene of a specific molecular weight. Another disadvantage is that concatemerization
of genes encoding large ELPs (>100 kDa) is challenging.
Another method, known as recursive directional ligation by plasmid reconstruction
(PRe-RDL), improves on the shortfalls associated with concatemerization(Hassouneh et
28
al., 2012). The mechanism for construction of these plasmids involves the use of Type
IIs restriction enzymes that cut to the 3’ direction of their recognition site. By exact
placement of recognition sites before the start codon and after the stop codon, these
enzymes can produce complementary sticky ends inside the ELP gene. Two batches of
such plasmids can be doubly digested with the unique type IIs enzyme and a second
enzyme found elsewhere on the plasmid. By gel purification, the two halves of the
plasmid, each containing the entire ELP gene, can be ligated together. This
reconstitutes the entire plasmid (origin of replication, antibiotic resistance, ribosome
binding site, etc.), while retaining only the restriction sites proximal to the start and stop
codon for the ELP. More importantly, the resulting gene now encodes an in-frame fusion
of both plasmids. This can be used to double the length of an ELP gene, or to graft
different genes that encode for block copolymers(Janib et al., 2014b). PRe-RDL can be
repeated in multiple iterations to generate larger plasmids and lends itself to the
eventual expression of entire ELP libraries with specific molecular weights. By use of
the Type IIs restriction enzymes, no restriction sites encode amino acids that are
expressed in the resulting protein polymer. As such, PRe-RDL is not limited to the
construction of ELP genes and can generate any protein polymer composed from
repetitive DNA sequences. Beyond PRe-RDL, other methods employed in the genesis
of functional ELPs include polymerase chain reaction (PCR)(Kurihara et al., 2005),
seamless cloning(McMillan et al., 1999), and overlap extension rolling circle
amplification (OERCA)(Amiram et al., 2013b; Amiram et al., 2011).
29
1.3.2. Protein expression and physicochemical characterization
ELPs have been commonly expressed in E. coli, owing to cost effectiveness and ease
of scale up, but expression from other organisms as varied as yeast(Sallach et al.,
2009), fungus(Herzog et al., 1997), and plants(Conley et al., 2009) has also been
detailed. In addition, ELPs exhibiting more elaborate architectures at the genetic level
have been designed such as the diblock copolymers pioneered by the laboratories of
Conticello(Wright and Conticello, 2002) and Chilkoti(Chilkoti et al., 2002). These protein
polymers exhibit self-assembly arising from the amphiphilic properties imparted by two
distinct blocks of differing polarities. This mediates partitioning of diblock copolymers
under aqueous conditions into spherical micelles that consist of a hydrophobic ELP core
and a hydrophilic ELP corona. The resulting nanoparticle is stable over a 5–10°C range
between the critical micelle temperature (CMT) of the hydrophobic block and the bulk
phase transition temperature mediated by the hydrophilic ELP corona. The hydrophobic
core itself may be employed in the encapsulation of hydrophobic drugs to increase their
solubility(Shi et al., 2013) while the hydrophilic corona can be imbued with functional
groups to enhance targeting(Dreher et al., 2008; Sun et al., 2011). Further increases in
temperature mediate formation of micron-sized aggregates more characteristic of the
bulk ELP phase separation.
In general, micelle assembly may improve a protein’s pharmacokinetics and
biodistribution since the usual micelle hydrodynamic radius (5 < Rh <50 nm) permits
30
evasion of the mononuclear phagocyte system and limits glomerular filtration(Malam et
al., 2009). Similar to monomer ELPs, altering the amino acid sequence, molecular
weight, and hydrophilic-to-hydrophobic ratio of the block copolymer will dictate CMT,
determine hydrodynamic radius, and yield control over micelle stability. As an example,
an ELP’s particular properties may be tuned to allow formation of a micelle under all
physiological temperatures for drug delivery purposes. It should further be noted that
triblock copolymers(Wright and Conticello, 2002), dendrimers(Kojima and Irie, 2013),
vesicles(Park and Champion, 2014), and chemically crosslinked hydrogels(Wang et al.,
2014a) have also been described.
ELP purification from E. coli lysate relies on the exploitation of an ELP’s previously
mentioned thermal responsiveness. This method is called inverse transition cycling
(ITC) and serves as a facile means of separating protein polymers from cellular debris
without the need for chromatography. In brief, after cell membrane disruption, the ELP
phase transition is triggered by heating above the Tt, allowing ELP coacervates to be
collected via centrifugation. Once the supernatant is removed, the ELP pellet is
resolubilized in cold buffer and centrifuged below the ELP Tt. This provides a means of
eliminating insoluble cellular debris and other contaminants captured in the pellet,
completing one cycle of ITC. Multiple ITC rounds effectively raise ELP purity to levels
above 95%, rendering the material suitable for study. Following purification, ELPs can
also be lyophilized for long-term storage. Useful workflows detailing this entire
purification process can be found in many general reviews of the ELP
31
literature(Hassouneh et al., 2012; MacEwan et al., 2014).
The versatility of these protein polymers is further underscored by the fact that ELP
sequences can be appended to other proteins as demonstrated in 1999 (Meyer and
Chilkoti, 1999), forming fusions at either the N-terminus or C-terminus capable of
imparting unique biological activities. Interestingly, one study elucidated that orientation
of the protein relative to the ELP played a large role in determining yields obtained of
the resulting fusion protein(Christensen et al., 2009). Localizing the ELP segment at the
C-terminal end of the target protein (protein-ELP) proved superior to N-terminal ELP
constructs (ELP-protein). At the most basic level, protein-ELP fusions can serve as
purification tags. These fusion proteins may also function as macromolecule carriers
aimed at increasing molecular weights to retard filtration by the kidneys. Diblock
copolymers have subsequently been manipulated to exploit the presence of peptide or
protein moieties on an ELP micelle corona and allow high-avidity uptake(Shi et al.,
2013; Simnick et al., 2010).
Small molecule drugs conjugated with ELPs, meanwhile, gain properties of thermally-
induced phase separation while retaining their bioactivity. For instance, one system
involved the chemical conjugation of an ELP to the chemotherapeutic,
doxorubicin(Dreher et al., 2003; MacKay et al., 2009). In another study, micelle
formation was exploited to present multivalent targeting motifs(Dreher et al., 2008),
which enhanced cellular uptake. ELPs have been utilized in tandem with recombinant
oligopeptide fusions involving cell-penetrating peptides(Massodi et al., 2005), a c-myc
32
oncogene inhibitor(Bidwell and Raucher, 2005), and recombinant protein fusions with
interleukin-1 receptor antagonist(Shamji et al., 2007). Surfaces coated with an ELP
fused to the RGD or fibronectin CS5 cell binding sequence retained the ability to
support in vitro endothelial cell adhesion and spreading(Liu et al., 2004). Additional uses
of ELPs include purification of holoenzymes such as D-Amino acid oxidase (Du et al.,
2015), isolation of multisubunit enzymes like RNA polymerase (Fong et al., 2010),
entrapment of small molecules(Shi et al., 2013), and the generation of ELPs hybridized
with other polymers (Wang et al., 2014a).
The genetically-encoded nature of ELPs permits the generation of monodisperse
biopolymers(Frandsen and Ghandehari, 2012). Considering that polymer molecular
weight plays a large role with regard to in vivo drug carrier disposition, ELP
monodispersity lends itself to the control of pharmacokinetic parameters such as
biodistribution and clearance rate. Other studies were integral in establishing the
foundations for using microPET imaging as a non-invasive means of tracking ELP
biodistribution and image-driven pharmacokinetics(Janib et al., 2013). This was
accomplished by conjugating a chelating agent, AmBaSar, onto the ELP prior to
radiolabeling with
64
Cu. In contrast to the classical method of obtaining blood samples to
deduce pharmacokinetics, this imaging modality allows for the application of image-
driven pharmacokinetic modeling(Qin et al., 2009). These studies confirmed that a low
molecular weight soluble ELP (<40 kDa) was cleared rapidly by the kidney, and that
high molecular weight soluble ELPs (>70 kDa) were retained in the blood for long
enough to generate an EPR-based enhancement in a breast cancer xenograft model.
33
Interestingly, a nanoparticle composed of an ELP diblock copolymer demonstrated
slightly enhanced hepatic clearance. An advantage afforded by microPET imaging
coupled with image-driven pharmacokinetics involves data acquisition that neither
compromises the physiological system under investigation nor suffers limitations
encountered when removing blood and radiolabeled agent from the subject.
As a biotherapeutic, ELPs have agreeable pharmacokinetics with terminal circulation
half-lives ranging from 8 to 12 hours in mice(Janib et al., 2013; Liu et al., 2006; MacKay
et al., 2009). Based on prior work in small animal models, ELPs also exhibit
biocompatibility and low immunogenicity(Cappello et al., 1998; Megeed et al., 2002;
Nouri et al., 2015). One study by Cho et al(Cho et al., 2015) involved the application of
immune-tolerant elastin-like polypeptides (iTEPs) as cytotoxic T-lymphocyte vaccine
carriers. iTEP design places an emphasis on humoral tolerance through the absence of
epitopes that can bind T and B cells. The motif selected to generate the repeats of
iTEPs A to D were sourced from the homologous peptide sequences of mouse and
human elastins. In addition, this 18 residue motif is longer than either MHC class II-
restricted TCR epitopes or linear BCR epitopes to further avoid immunogenicity. Despite
lacking the canonical pentapeptide sequences, iTEPs still retain phase transition
properties. Each of the four iTEPs elicited negligible iTEP-specific antibody titers
relative to an ovalbumin (OVA) control. Another interesting finding was the fact that in
vivo aggregation of hydrophobic constructs (iTEPs A, C, and D) did not influence
immunogenicity in any manner differing from soluble forms (iTEP B). At the biophysical
level, this negligible immunogenicity may originate from the high energy levels required
34
for host antibodies to overcome when
binding to the predominantly random
coiled structure of solvated ELPs at
potential epitopes(Meyer et al., 2001; Urry
and Parker, 2002; Urry et al., 2010). In
combination with their lack of positive or
negative charge, limited number of
primary epitopes, and the similarity of their
pentameric repeat to short fragments of
human tropoelastin, the possibility
remains that they have a low likelihood of
producing an adaptive immune response.
This particular interpretation has been
borne out by data from PhaseBio Inc,
which suggest that ELPs are safe in
human clinical trials(Pharmaceutics,
2012).
It should be expected that the biological
composition of these protein polymers
mediates their biodegradation into
peptides and amino acids readily removed from the body. One in vitro study by our
laboratory(Shah et al., 2012) demonstrated that transformed PTEN-deficient mouse
hepatocytes enzymatically degrade ELP nanoparticles (Figure 4). The kinetics of
Figure 4. Uptake and degradation of
ELP nanoparticles. Rhodamine-
conjugated S48I48 (red) was
incubated with transformed murine
hepatocytes for 0, 2, 4, 24 h at 37°C
and imaged using live-cell confocal
fluorescence microscopy. Cells were
counterstained with lysotracker green
to show low pH compartments
associated with lysosomal protease
activity. Differential interference
contrast (DIC) imaging illustrated cell
morphology. ELP nanoparticles were
visible within and on the cell surface
after 2 h; intracellular staining
significantly decreased after 24-h
incubation. The arrow indicates
internalized nanoparticles with the
lysosomes. Scale bar: 10 μm.
Reprinted from Shah et al., 2012 with
permission of John Wiley and Sons.
35
biodegradation have also been analyzed in vivo using a 59.4 kDa
14
C-labeled ELP. In
that study, intravenous administration led to a relatively modest degradation rate of ~2.5
wt % per day, pointing to the fact that ELPs can achieve in vivo stability for therapeutic
applications(Liu et al., 2006).
There have been reports that ELPs do not significantly affect whole blood clotting time
in dogs or induce red blood cell hemolysis in rabbits(Mackay and Chilkoti, 2008).
Though all ELPs are eventually subject to clearance, ELP solubility ultimately affects
these degradation kinetics. Ex vivo experiments deduced that soluble ELPs below Tt
experience enzymatic degradation by elastases as well as collagenases. The formation
of coacervates above an ELP transition temperature partially blocked proteolysis by
collagenase. In contrast, an elastase degrades both soluble and coacervate ELP(Shah
et al., 2012). While there may be conditions where ELP coacervation is protective
against a proteolytic environment, this appears to vary case-by-case.
In the design of next generation, clinically-relevant nanomedicines, ELPs have
demonstrated evidence supporting the following advantages:
i) Genetically-engineered precision—as the amino acid sequences of ELPs can be
tailored at the genetic level, their biosynthesis affords excellent control over
architecture, rendering monodisperse polymers from which to build multifunctional
therapeutics. ii) Safety—ELPs appear to be biocompatible, biodegradable, and non-
immunogenic. iii) Environmentally-responsiveness—the phase behavior of the ELP can
36
be tailored to assemble multivalent nanoparticles in aqueous solution without solvents
or crosslinkers, while maintaining the activity of fusion proteins.
1.4. Applications of Elastin-like Polypeptides
The versatility of ELPs as nanomedicines is driving interest in their adoption across a
broad spectrum of disciplines as depicted in Figure 5. Anti-cancer strategies rank chief
among their applications, with oncology capturing about 35% of total market revenue in
the nanomedicine
sector(Pillai, 2014; Wang et
al., 2013). Beyond
oncology, however, there
exists an exciting landscape
of pioneering methods
involving ELPs in fields as
diverse as gene delivery,
ophthalmology, and
maternal-fetal medicine.
What follows is a catalogue of these various applications as they pertain to translational
approaches and the potential for eventual commercialization.
Figure 5. The various applications of ELPs as
nanomedicines
37
1.4.1. Cancer
According to the American Cancer Society, over 1.7 million new cases of cancer will be
diagnosed in 2018(Siegel et al., 2018). Though currently the second leading cause of
death in the United States, it is expected to surpass heart diseases in coming years.
This has prompted researchers to accelerate innovations in anti-cancer nanomedicine,
and ELPs are one emerging tool in the field. Since the breadth of ELP cancer research
extends beyond the purview of this article, only a few instructive examples will be
detailed.
One of the first cancer-based applications was performed by Dr. Chilkoti and
colleagues, who demonstrated that hyperthermia could enhance tumor localization of
ELPs in SKOV-3 ovarian carcinoma and D-54MG glioma cell lines(Meyer et al., 2001).
This link was further solidified between mild hyperthermia and tumor penetration by
studying the sequestering of
14
C-radiolabeled ELPs using FaDu tumor grafts in nude
mice models(Liu et al., 2006). For a soluble ELP (Tt = 40°C), a terminal plasma half-life
approaching 8.7 hours was achieved. Once the tumors were heated to 42°C, the
thermally responsive [
14
C]ELP1 exhibited a 1.5 fold increase in accumulation rates over
a thermally unresponsive [
14
C]ELP2 control.
Conjugation of small molecule drugs to ELPs represented another early advance aimed
at decreasing toxicities and enhancing therapeutic efficacy. In one system, an N-
terminal lysine present on the ~60 kDa ELP1-150 was conjugated to a pH-sensitive
hydrazone bond(Dreher et al., 2003). This bond mediated attachment of a maleimide
38
linker ferrying doxorubicin, an anticancer agent which acts through inhibition of
topoisomerase. Following endosomal uptake in FaDu cells, the pH responsive ELP-
hydrazone portion is severed in the acidic lysosome, allowing free doxorubicin to reach
the nucleus and effect its cytotoxicity. Interestingly, in vivo studies revealed similar
cytotoxicity following administration of both free doxorubicin and doxorubicin-ELP
despite the differing intracellular distribution rates. Modifications to the length and
structure of the doxorubicin-ELP maleimide linker offers an additional means of tuning
aspects of the release profile without dramatically altering ELP Tt. Most prominently, it
was deduced that shorter linkers between doxorubicin-ELP conjugates enhanced drug
release(Furgeson et al., 2006). This approach was further refined to deliver up to 8
doxorubicin molecules covalently linked to one end of the ELP, which promoted the
assembly of ELP-coated nanoparticles (MacKay et al., 2009). These chimeric
polypeptides were the first ELPs reported to block tumor growth in vivo; furthermore,
they appeared to be effective after only a single dose.
Building on those studies, a research team based at the Samsung Advanced Institute of
Technology led by Kim and colleagues took a hybrid approach by merging ELPs with
other drug delivery technologies(Park et al., 2014). Temperature-sensitive liposomes
were generated with DPPC/Chol/DSPE-PEG serving as the primary lipids, which
incorporated a stearyl group appended to the amino terminus of an ELP via an amide
bond called SA-V3. Doxorubicin was encapsulated inside the resulting ELP liposomal
formulation at a ratio of 1:0.2 (w/w, phospholipid:doxorubicin). Most notably, over 80%
of the encapsulated drug exhibited release within 5 minutes following a temperature
39
increase to 42°C. Liposomes lacking SA-V3, by contrast, experienced doxorubicin
release rates of less than 10% after administration of mild hyperthermia. Six-week old
male BALB/c nude mice were later used to implant tumors deriving from EMT-6, a
murine mammary cell line. Overall stable blood circulation times were observed with the
in vivo half-life of encapsulated doxorubicin persisting for about 2.5 hours. Finally, a
study was conducted to determine drug accumulation after preheating a tumor for 30
minutes at 42°C. ELP liposomes displayed significantly better tumor localization at 6
and 12 hours after preheating; furthermore, these levels exceeded the concentration
achieved for free drug by 31-fold. Additional hybridized systems, including PEG-
functionalized ELPs(van Eldijk et al., 2014), are also currently under investigation by
several other groups.
Alternative strategies have focused on designing protein polymers capable of
overcoming transport barriers, such as the P-glycoprotein efflux pump (Bidwell and
Raucher, 2010; Massodi et al., 2005; Ryu and Raucher, 2015). Bidwell and Raucher
validated this approach using a cell-penetrating peptide (CPP) appended to an ELP in
MCF-7 breast carcinoma cells(Bidwell and Raucher, 2005). In this study, penetratin
(AntP), a 16 amino acid sequence capable of endocytic uptake, was designed in
tandem with a peptide (H1) inhibitor of the c-Myc proto-oncogene’s transcriptional
activities. In vitro aggregation at 42°C, mediated by the temperature-sensitive AntP-
ELP-H1, enhanced cell death 2-fold versus controls containing non-heated cells or
thermally unresponsive ELP. Further advances have led to many derivations and in vivo
applications of the original CPP-ELP concept in combination with peptides as varied as
40
Bac(Ryu and Raucher, 2014b), p21(Ryu and Raucher, 2014a; Walker et al., 2014), and
Tat(Massodi et al., 2009) against neoplasms. Moreover, research into the uptake
mechanisms of CPP-ELPs deduced via flow cytometry that endocytosis of these
polymers occurs in a caveolae-independent manner(Bidwell and Raucher, 2010).
Other tumor-targeting modalities have also been described in ELP-related literature.
One topic explored in the MacKay laboratory targets angiogenesis. Unregulated blood
vessel growth remains a stable hallmark of many solid tumors which require access to
nutrients in the bloodstream. Such neovasculature differentially overexpresses integrin
αV heterodimers and can thus be targeted by RGD-containing peptides(Boudreau and
Varner, 2004; Reiss et al., 2006). Certain antagonists, most prominently disintegrins
derived from viper venoms, offer a natural source of high affinity ligands binding these
proteins. While contortrostatin (CN) is a prototypal member of the disintegrin family
obtained directly from venom(Minea et al., 2005), recombinant approaches have since
been employed by Markland and colleagues to generate vicrostatin (VCN) by bacterial
fermentation, which have been evaluated in murine models of breast and prostate
cancer(Minea et al., 2010).
Fusion of VCN to a ~73 kDa ELP sequence G(VPGAG)192Y, also known as A192,
formed spherical multimers while retaining VCN’s integrin-binding and receptor-
mediated uptake capacities as demonstrated on HUVECs(Janib et al., 2014a). When
compared to a control ELP fused to a linear RGD segment, the VCN-A192 construct
outperformed with a 30-fold lower IC50 in vitro. The higher molecular weight of VCN-
41
A192 also solves a pressing challenge inherent in the use of free VCN— namely, the
rapid clearance of this small peptide therapeutic via renal filtration. An orthotopic murine
model of breast carcinoma demonstrated that VCN-A192 not only slowed clearance but
also promoted tumor accumulation relative to either A192 or VCN alone.
Parallel in scope to those efforts, the Chilkoti laboratory established a means for
controllable tumor targeting using micelle-forming ELP diblock copolymers. This
rationale arises from the fact that, while high affinity nanoparticles dictate enhanced
accumulation at target sites, high affinity may introduce toxicities involving healthy
tissues expressing the same receptor at reduced levels. Solving this issue necessitates
development of a construct with a low affinity for receptors in healthy tissue that shift to
a high avidity form in response to a trigger— such as the application of heat. To that
end, the GRGDS peptide targeting the αVβ3 integrin was fused to an ELP diblock
copolymer(Simnick et al., 2010). While slight binding was demonstrated in the
monomeric form, above the critical micelle temperature these peptide-decorated
nanoparticles drove high-avidity uptake by endothelial cells. More recent studies have
focused on a RGD-TRAIL-ELP and its apoptotic activity in both human colorectal
carcinoma (COLO-205) and human breast cancer (MDA-MB-231) cell lines(Huang et
al., 2015).
Subsequent studies have expanded the use of ELP diblock copolymers with data
indicating that larger proteins, consisting of >100 residues, fused to diblock ELP
segments similarly preserve biological moieties and their functions without abolishing
42
ELP micelle formation. A significant set of experiments (Shi et al., 2013) validated this
ELP drug delivery strategy using a hydrophobic, immunosuppressive macrolide derived
from Streptomyces hygroscopicus known as rapamycin. In contrast to direct conjugation
of small molecules onto a polymer platform, this novel departure utilizes high affinity
binding between rapamycin and its cognate human receptor, FKBP12 (KD=0.2 nM).
FKBP was fused to an ELP diblock copolymer, which presented FKBP on the corona of
a resulting nanoparticle stable at physiological temperatures. Following passive uptake,
biodegradation, and drug release, freed rapamycin binds to native FKBP and inhibits
mTOR-mediated cell cycling (Figure 6A). mTOR is a serine/threonine kinase bridging
upstream signal inputs (e.g. PI3K/AKT pathway) with downstream effectors through
signaling cascades and is thus commonly exploited during tumorigenesis(Banaszynski
et al., 2005; Yuan et al., 2009). While the anti-proliferative properties of rapamycin have
been found in neoplasms of the breast, prostate, and colon, the free drug’s utility is
tempered by low solubility, low bioavailability, and dose-limiting side effects in the lungs,
kidneys, and liver. ELP diblock copolymers exhibiting high avidity for rapamycin were
designed to prevent filtration/accumulation in the kidney and permeability into normal
tissues, raise its poor solubility, and increase the tolerated dose.
43
Fusion between FKBP and a 39 kDa diblock copolymer ELP (SI) self-assembles into a
stable nanoparticle (FSI) with a 24 nm hydrodynamic radius and a CMT of 24.5°C.
Relative to plain SI, the hydrodynamic radius and stability of FSI was nearly identical
(Figure 6B). A novel two-phase
solvent evaporation method had
to be adapted to facilitate
encapsulation of rapamycin into
FSI. Both TEM and DLS
confirmed that the radii of FSI
nanoparticles following drug
encapsulation were only slightly
larger than the preceding
values.
The FSI release half-life was
explored via dialysis under sink
conditions, which revealed a
biexponential release profile,
with a fast phase (1.9 hr half-
life) followed by a slow (57.8 hr
half-life) terminal phase. This
biphasic behavior is illustrative
of the fact that FSI not only encapsulates a reservoir of rapamycin within its hydrophobic
Figure 6. Design of Elastin-like polypeptide
(ELP) nanoparticles that carry an anti-
proliferative drug at both their core and corona.
(A) High-avidity interaction between a small
molecule drug (rapamycin) and its cognate target
protein (FKBP) decorated at surface of an ELP
nanoparticle. The nanoparticles assemble
nanoparticles above a critical micelle temperature
(CMT). (B) Dynamic light scattering of FKBP-
decorated FSI and plain SI nanoparticles shows
that protein modification minimally affects CMT or
hydrodynamic radius. (C) Tumor growth inhibition
by FSI-rapamycin versus free rapamycin (0.75
mg/kg BW). Free rapamycin mice were sacrificed
at Day 24 due to toxicity. Reprinted from Shi et al.,
2013 with permission of Elsevier.
44
core (i.e. rapid release) but also retains approximately 30% of the drug on FKBP
decorating the micelle corona. To confirm this mechanism, rapamycin was solubilized
within the unmodified diblock copolymer SI alone, which released all of its contents with
a 2.2 hr half-life, similar to FSI’s initial release phase. In another manuscript, it was
demonstrated that free FKBP-ELP released drug under sink conditions with a
monoexponential decay and a half-life of just 13 hours(Shah et al., 2013a). Together,
these data suggest that by coassembly of multiple copies of FKBP at the surface of a
nanoparticle, it may be possible to cooperatively drive avidity for target small molecules.
This observation could have many potential uses; however, in the case of FSI, it
appears to promote long duration release of rapamycin from the carrier.
In vitro cell proliferation assays confirmed rapamycin-bound FSI (IC50=0.28 nM) was just
as effective as free drug (IC50=0.27 nM) in reducing the viability of MDA-MB-468 breast
cancer cells sensitive to the drug. In contrast, neither FSI-bound nor free drug affected
the rapamycin-insensitive MDA-MB-231 cell line. This FSI formulation also extended
drug solubility at least 10-fold above that of the free drug. Tumor regression studies
conducted in orthotopic breast cancer xenografts led to the premature withdrawal of
mice committed to dosing regimens with free rapamycin due to cumulative toxicities.
FSI, on the other hand, surpassed its free counterpart by mitigating drug toxicities
without sacrificing cytostatic efficacy (Figure 6C).
Another notable anti-cancer strategy revolves around merging ELP technology and
antibody therapeutics. Antibodies have figured prominently in the world of biotechnology
45
with the first therapeutic mAb (muromonab-CD3 for prevention of acute transplant
rejection) approved by the FDA in 1986(Ecker et al., 2015). Rituximab, a chimeric mAb
capable of binding CD20 to induce apoptosis in B-cell malignancies, constitutes one
example which has since been deemed an essential medicine by the World Health
Organization(Sousou and Friedberg, 2010). While single chain variable fragments
(scFv) have been proposed as a versatile class of antibody therapeutics(Holliger and
Hudson, 2005), their small size leads to poor tumor retention and rapid clearance (~4
hrs). To address these shortcomings, many investigators have turned to bioconjugation
with high molecular weight polymers as a means of prolonging scFv half-lives(Johnson
et al., 2009). Applying those findings to ELPs, anti-CD20 scFv was generated through
fusion to the ~73 kDa A192 monomer by Aluri and coworkers(Aluri et al., 2014).
Dynamic light scattering, phase diagrams, and transmission electron microscopy
revealed that anti-CD20 scFv-ELPs unexpectedly formed nanoworm structures with a
hydrodynamic radius of 85.7 ± 16.5 nm. In contrast, the unmodified A192 ELP exhibited
a more typical value of 6.7 ± 0.2 nm. As the A192 ELP is not a diblock copolymer, its
simple addition provides no mechanism for particle assembly. Instead, it appears that
particle assembly is mediated by the scFv domain. scFv are frequently challenging to
produce and stabilize; furthermore, a likely explanation for the nanoassembly of the
scFv-ELP is that the scFv domains partially aggregate to form the core of a particle held
together by noncovalent intermolecular associations. Instead of flocculating, the high
molecular weight A192 protein polymer sterically stabilizes these colloids. More
surprising, in vitro studies confirmed binding of CD20 receptor and efficient induction of
46
apoptosis by the nanoworms. Viability assays revealed an IC50=32 μM in Raji cells and
IC50=41 μM for SU-DHL-7 B-cell lines. Tumor regression was examined using Raji
xenografs in athymic nude mice. Median survival times for the ELP nanoworms were
significantly longer than for monoclonal Rituximab or PBS. Notably, upon cessation of
therapy, these tumors continued to proliferate. Nevertheless, the surprising stabilization
of these nanostructures and the finding that they maintain efficacy suggests this class of
nanoparticles may be suitable for additional study.
1.4.2. Ocular diseases
With the National Eye Institute reporting an economic burden for eye diseases
surpassing 139 billion USD in 2014, ocular therapeutics represent another urgent
growth opportunity(Institute). In particular, novel ELP-based solutions have been
devised to combat corneal wounds(Haddadin et al., 2013) and Sjögren’s syndrome.
Each year, about 2.4 million eye injuries occur in the US(Ophthalmology), some of
which are exacerbated into sight-threatening conditions.
The discovery of lacritin(Karnati et al., 2013), a mitogen found in tears promoting
survival of corneal epithelial cells, provided a compelling rationale for the design of a
lacritin-ELP fusion (LSI) exhibiting multivalent presentation to treat ocular abrasion
wounds (Wang et al., 2014b). This fusion was composed of the lacritin domain
appended to the ELP diblock copolymer discussed above (SI), which by itself
assembles nanoparticles (Rh ~ 25 nm) above 25°C. A shift in particle diameter at a
temperature of 18.4°C was observed for LSI, but interestingly, the construct
47
preassembled into 30-40 nm
nanoparticles even below this
transition temperature. Larger LSI
nanoparticles approaching 140 nm
were subsequently isolated at
physiological temperatures. To
assay LSI’s mitogenic activity in
vitro, a scratch was applied to
confluent human corneal epithelial
cells and the healing process was
observed over 24 hrs. At
concentrations as low as 10 nM,
LSI accelerated scratch wound
healing in a manner comparable to
a positive control containing
epidermal growth factor (EGF) and
bovine pituitary extract (BPE). In
vivo efficacy studies were then
investigated via induction of
circular defect in the corneal
epithelium in NOD mice (Figure 7A). Mice were segregated into four groups— KSFM
media supplement with BPE (50 µg ml
-1
) + EGF 5 ng ml
-1
, LSI (100 µM), plain SI (100
µM), or no treatment— then allowed to heal for 24 hrs following two rounds of topical
Figure 7. Lacritin-ELP nanoparticles heal
corneal wounds in mice. A 2 mm defect in the
corneal epithelium of female non-obese diabetic
(NOD) mice was monitored using fluorescein
staining at 0, 12 and 24 h with or without
treatment by LSI, SI, and a positive control EGF +
BPE. (A) Representative images showing the
time-lapse healing of the corneal wound. (B) LSI
at both 12 and 24 h significantly (***p = 0.001, n =
4) decreased the percentage of initial wound area
(PctArea) compared to SI, EGF + BPE, and no
treatment groups. (C) After 24 h, corneas were
fixed, sectioned across the defect, and stained by
hematoxylin and eosin. The corneal epithelium of
the LSI treatment group revealed normal
pathology. Although reduced fluorescein staining
was observed at late times in the SI group, the
epithelium did not recover fully, as evidenced by
its irregular surface (black arrows). Reprinted from
[124] with permission of The Royal Society of
Chemistry.
48
administration. Fluorescein staining and percentage of initial wound area values both
confirmed that treatment with LSI outperformed SI, BPE + EGF co-treatment, and no
treatment controls (Figure 7B).
These findings were corroborated through histological analyses showing improved
morphology of the corneal epithelia after LSI-mediated healing (Figure 7C). Follow-up
LSI studies further emphasized the critical contribution of multivalency to this efficacy. In
comparison to a thermally-insensitive ELP control (LS96), LSI induced more profound
corneal wound healing over a 12 hr timeframe. More recent data indicates that Lacritin-
ELPs are also capable of phase separating in the lacrimal gland, where they mimic the
functional effects of free lacritin by promoting tear protein exocytosis (Wang et al.,
2015).
The autoimmune disease known as Sjögren’s syndrome (SjS) affects approximately 4
million patients with 9 out of 10 being women(Patel and Shahane, 2014). The primary
symptoms include debilitating dry eye and dry mouth. The MacKay group, in
collaboration with the Hamm-Alvarez laboratory, therefore sought to further adapt the
FSI rapamycin nanoformulation discussed earlier to encompass ocular indications(Shah
et al., 2013a). This rationale arises from the fact that, while this small molecule drug
acts as an immunosuppressant, it has never been investigated as a means of treating
the severe autoimmune inflammation of the lacrimal gland, called dacryoadenitis, using
the NOD model that replicates some of the pathology found in SjS. The
physicochemical characterizations and release profiles of FSI were consistent with the
49
earlier breast cancer study.
The non-obese diabetic (NOD) mouse model was selected for in vivo work owing to its
recapitulation of symptoms, including autoimmune-mediated lymphocytic infiltration of
lacrimal gland, characteristic changes in the spectrum of tear proteins secreted, and
development of serum autoantibodies, regularly seen in human SjS. FSI, free drug,
PBS, and untreated control groups were assessed for their efficacy in reducing
lymphocytic infiltration of the lacrimal gland using image analysis of the percent area
infiltrated by lymphocytes. FSI exhibited a ~50% reduction in infiltration area following
only 3 injections in one week. Free rapamycin likewise displayed a similar decrease
when examined relative to both PBS and untreated mice; however, this efficacy was
accompanied by histopathology consistent with renal toxicity. Nephrotoxicity was
observed for free rapamycin, which exhibited a greater degree of kidney tubule
vacuolization relative to FSI-encapsulated drug. Toxicological studies involving FSI-
bound versus free drug showed that the former was far better tolerated. Only 4% of FSI-
treated mice, for instance, had bruised tails post-injection. Injections with the free drug
conversely led to a 70% increase in edematous tails with 26% becoming bruised and
4% ending up necrotic. When lacrimal gland gene expression was assessed by qPCR,
the FSI-encapsulated drug shifted transcriptional patterns for a panel of genes
associated with the mTOR pathway in comparison to a similar dose of the free drug.
Beyond simply adjusting the toxicity profile for rapamycin, these data suggest that
carriers like FSI may also play a large role in the delivery of the drug to diseased target
tissue, modulating a change in disease pathology. ELP constructs, such as FSI and
50
others, may have broader utility in other autoimmune and inflammatory disease
conditions.
1.4.3. Cardiovascular diseases
Cardiovascular diseases (CVDs) mark the primary cause of death worldwide with
600,000 fatalities reported each year in the US alone. Similarly sobering is the fact that
CVD accounts for 313 billion USD in costs due to health expenditures and lost
productivity(Healthline). Among the risk factors for CVD, hypertension remains most
amenable to pharmacological modulation as indicated by the broad range of
antihypertensive medications on the market(Oparil and Schmieder, 2015). PhaseBio
Inc. has pioneered an ELP-based treatment for essential hypertension using a VIP-ELP
fusion protein known as Vasomera™. Vasoactive intestinal peptide (VIP) is a
neuropeptide, consisting of 28 amino acid residues, which acts as a ligand of the G-
protein coupled receptors VPAC1 and VPAC2(Dvorakova et al., 2014). As VIP
promotes heart contractility and induces coronary vasodilation, therapeutic applications
against hypertension provide a compelling rationale for its exploitation.
Fusion of ELP to an analogue of VIP specific for VPAC2 effectively produced a long-
acting biotherapeutic, exceeding the brief half-life (<2 min) of the parent peptide, due to
the greater molecular weight imparted by the ELP segment(Yeh, 2012). To deduce the
VIP-ELP’s effect on blood pressure, SHR rats were induced to develop pulmonary
arterial hypertension and then received either VIP-ELP (3-6 mg/kg) or placebo (0.9%
NaCl) delivered via single bolus doses intravenously or intratracheally. This led to rapid
51
reductions in pulmonary artery pressure (-24 ± 3% from 41 ± 1 to 31 ± 1 mmHg) and
sustained (>5 min) vasorelaxation independent of administrate route (IV: -17 ± 4%
versus IT: -28 ± 4%). Long-term hemodynamic studies were conducted in conscious
rats where VIP-ELPs mediated dose-dependent blood pressure decreases that were
sustainable for about 12 hours post-dosing.
VIP-ELP was subsequently investigated for its potential in mitigating heart failure using
rat models of doxorubicin-induced cardiomyopathy(del Rio et al., 2012). Daily
intravenous administration of VIP-ELPs combated myocardial dysfunction and
prevented muscle wasting. These protein polymers also reduced the energetic
demands of diseased cardiac tissue while simultaneously improving left ventricular
systolic/diastolic functioning. When canines were given the VIP-ELPs, both groups with
healthy and failing hearts exhibited a dose-dependent increases in cardiac function
within the 0.1 to 1 µg/kg/min range(del Rio et al., 2013).
Those precedents established the foundation for Phase I single ascending dose trials by
PhaseBio in patients with pulmonary arterial hypertension(Pharmaceutics, 2013).
Introduced through once weekly dosing regimens, VIP-ELPs were found to be safe and
well-tolerated by subcutaneous and intravenous routes. They also demonstrated a
prolonged, dose-dependent reduction in blood pressure. Recent work now focuses on
treating the cardiac dysfunction associated with both Duchenne and Becker muscular
dystrophy. This novel biotherapeutic has since been awarded Orphan Designation
status by the FDA.
52
1.4.4. Maternal-fetal medicine
Pregnant women have largely been overlooked as a patient population by the
pharmaceutical industry owing to the fear of formulating an agent that, while beneficial
to the mother, may pose risks to the fetus(Bidwell and George, 2014). Drug
development for pregnancy-related disorders has therefore been a slow process.
Preeclampsia, also known as pregnancy-induced hypertension, is one such disorder
characterized by elevated blood pressure, swelling, and proteinuria. Left undetected,
this condition can escalate to eclampsia which threatens both mother and child; one
estimate indicates this causes 40 to 60% of all maternal deaths in developing
countries(Development). The only effective treatment involves induction of delivery.
Encouraging breakthroughs by Bidwell and colleagues, however, suggest a novel
solution to the problems of maternal drug delivery from his earlier work using SynB1-
ELP, a cell penetrating peptide fusion(George et al., 2014). Pregnant Sprague Dawley
rats were given a bolus dose (100 mg/kg) intravenously to acquire pharmacokinetics
data between an ELP control and SynB1-ELP. While plain ELPs possessed a higher
initial plasma concentration (3,168 µg/mL) relative to SynB1-ELP (2,376 µg/mL), the
latter exhibited faster extravasation; this indicates that the SynB1 moiety mediated
quicker sequestering into tissue. After this rapid distribution in the first 60 minutes, a
slower terminal half-life prevailed with SynB1-ELP terminal half-life lasting 190.2 min in
contrast to 77.5 min for ELP alone.
53
Four hours after the bolus dose, tissues were removed and examined ex vivo using
whole organ fluorescence imaging to deduce factors such as biodistribution, fetal
uptake, and placental deposition. It was observed that ELP and SynB1-ELP constructs
accumulated at significant amounts in placental tissue, although only minute levels were
detected in the fetuses. Microscopic examination of placental tissues using a cytokeratin
counterstain, meanwhile, confirmed that ELP existed within trophoblast cell cytoplasm
yet remained excluded from the fetal side of the chorionic villi as intended. The effects
of a 5 day continuous infusion of the two ELP groups was subsequently explored.
Chronic infusion had a moderating effect on the biodistribution of SynB1-ELP when
compared to ELP. In other words, the tissue levels of the two achieved similar values.
Beyond this difference, results were comparable to those identified following a bolus
dose. Fetal tissue invasion by either of the ELP groups was negligible.
1.4.5. Diabetes
Diabetes describes a cluster of chronic metabolic diseases, involving high blood
glucose levels and insulin insufficiency, which affects more than 300 million people
worldwide; the CDC further predicts that a staggering 1 in 3 Americans could be
stricken by 2050(Prevention.). Within this patient population, type 2 diabetes essentially
amounts to 90 to 95 percent of all cases diagnosed in adults. One striking study
pursued focused on fusion of exenatide, a therapeutic analogue of glucagon-like
peptide 1 improving glycemic control, to the recombinant polypeptide known as
XTEN(Schellenberger et al., 2009). Based on allometric scaling in various animal
models, this fusion protein was extrapolated to enhance the peptide’s plasma half-life in
54
humans from 2.4 hrs to 139 hrs, which could potentially enable once monthly
administration.
These results provided suitable grounds for ELP experts to expand into new lines of
investigation. More specifically, the 30 amino acid glucagon-like peptide 1 (GLP-1),
which possesses a short half-life (<2 min), was chosen as an ELP fusion candidate due
to its role in promoting insulin release from pancreatic β-cells(Amiram et al., 2013a).
Two 50 kDa (GLP-1)-ELPs were designed with the first property of room temperature
solubility and the ability to form a stable, coacervate-based drug depot following
subcutaneous injection into C57BL/6J mice; the second fusion served as a control
which remained soluble at body temperature. Both constructs were examined in vitro for
stability against neutral endopeptidase, a protease known to degrade GLP-1.
Surprisingly, neither the soluble or depot-forming ELPs experienced degradation even
after 18 hr incubation with the enzyme at 20 or 37°C. In contrast, the native GLP-1 was
almost completely degraded. This underpins the fact that the increased molecular
55
weight heightened GLP-1 stability and could be considered in the formulation of longer-
acting therapies. GLP-1, however, still proved more potent (EC50=0.113 nM) in binding
GLP-1 receptor versus depot-capable (EC50= 4.75 nM) and soluble (EC50= 5.59 nM)
configurations.
According to biodistribution studies
determined by NIR fluorescence
tomography (Figure 8), the GLP-1
depot was retained at its injection site
for over 120 hours, while the soluble
GLP-1 formulation disappeared 24
hours post-injection. In vivo efficacy
data later demonstrated that (GLP-1)-
ELP formulation steadily reduced
blood glucose levels up to ~30% in a
dose-dependent manner (e.g. 175,
350, and 700 nmol/kg over 24, 72, and
144 hrs respectively). Alternatively,
the soluble (GLP-1)-ELP rapidly
precipitated a 60% glucose level decline at a dose of 175 nmol/kg, causing a peak and
valley response indicative of more rapid onset and loss of activity. These results affirm
that GLP-1 ELP fusions can reduce blood glucose levels across a range of doses at
intervals that depend on the phase behavior of the ELP. Based on these exciting
Figure 8. NIR tomography images of 175
nmol/kg (GLP-1)-ELPSol, 175 nmol/kg
(GLP-1)-ELPDepot, and 700 nmol/kg
(GLP-1)-ELPDepot at 0, 24, 72 and 120 hr
after subcutaneous injection. Reprinted
from Amiram et al., 2013a with permission
of Elsevier.
56
findings, (GLP-1)-ELP underwent Phase IIB trials as listed in Table 1 under the trade
name Glymera
TM
for once weekly treatment of hyperglycemia associated with type 2
diabetes(Pharmaceutics, 2012). Additional work has been conducted with a proinsulin-
ELP formulation, capable of being converted into mature insulin following subcutaneous
administration, alongside novel coformulations involving (GLP-1)-ELP.
Table 1. Elastin-like polypeptide (ELP) nanomedicines under commercial investigation.
Product/Agent Indications Status Company References
siEVI1-ELP Various cancers
(breast, ovarian,
pancreatic, and
lung)
Preclinical PeptiMed (Primiano,
2015)
Liposome-ELP
conjugates
Breast cancer Preclinical Samsung (Park et al.,
2014)
VIP-ELP
(Vasomera
TM
)
Pulmonary arterial
hypertension,
cardiomyopathies,
cystic fibrosis
Phase I PhaseBio (del Rio et al.,
2013; del Rio
et al., 2012;
Pharmaceutics,
2013, 2015;
Yeh, 2012;
Youngblood
BL, 2012)
GLP1-ELP
(Glymera
TM
)
Type II diabetes Phase IIB PhaseBio (Amiram et al.,
2013a; Amiram
et al., 2013b;
Pharmaceutics,
2012)
Another prominent area in diabetes management involves treating chronic wounds such
as pressure sores and foot ulcers. In the most extreme cases, amputations are
performed to stem tissue damage. With the advent of biotechnology, however, unique
solutions have emerged in the form of recombinant growth factors aimed at facilitating
wound re-epithelialization. Exogenous keratinocyte growth factor (KGF) is a monomeric
57
peptide shown to augment healing within wounds of complete and partial thickness in
animal models(Werner, 1998). Since KGF is administered topically, its activity and
bioavailability are both attenuated, necessitating large quantities to evoke the desired
clinical outcomes.
A KGF-ELP fusion was generated as a means of improving KGF efficacy (Koria et al.,
2011). Chief among their findings was the fact that KGF-ELP formed into 500 nm
nanoparticles despite the ELP segment having been designed linearly rather than as a
diblock copolymer. These results are not unexpected, however, considering that several
ELP fusions discussed previously have displayed similar behaviors(Aluri et al., 2014;
Wang et al., 2014b; Wang et al., 2014c). Seeking to determine whether KGF-ELP could
act on its receptor (KGFR), proliferation assays in A431 epithelial cells were performed.
Despite the apparent involvement in the KGF domain in mediating particle assembly,
proliferation rates were comparable for KGF-ELP and exogenous KGF (2.31-fold versus
2.0-fold respectively). A control ELP alone did not produce this growth response.
Another encouraging observation was the fact that KGF-ELPs phosphorylated
downstream effectors (e.g. ERK) of the receptor although at diminished levels relative to
KGF. Finally, in vivo studies were conducted employing genetically diabetic male mice
due to retarded wound healing. Treatments between KGF-ELP and free KGF revealed
that the former induced slightly more prominent re-epithelialization (36% coverage
versus 31% coverage respectively).
58
1.4.6. Regenerative medicine
Regenerative medicine seeks to restore lost functionality and promote self-healing of
damaged tissues. Regarding applications of the ELP platform, the Heilshorn group
pioneered a single-step process for rapid yet reproducible peptide conjugation coupled
with crosslinking of ELP hydrogels(Cai et al., 2014). In this case, a 15 amino acid
peptide (QK) capable of augmented binding and stability relative to vascular endothelial
growth factor (VEGF) was selected for ELP fusion. 2D cell proliferation studies using
HUVECs determined that QK-ELPs recapitulated proliferation at both 10 nM and 1 μM
relative to soluble QK in unmodified ELP hydrogels. Further work revealed a 10-fold
increase in the normalized protrusion area of HUVEC spheroids, defined as a ratio of
the cross-sectional area of protruding cells in the xy plane to the cross-sectional area of
the spheroid itself, within a 3D hydrogel system.
ELP depot formulations yield another means of combating certain ailments including
osteoarthritis due to the avascular nature of diseased sites. Intra-articular injections of
14
C-labeled ELP depots in rat models led to sustained release with an 85 hr half-life. In
dramatic contrast, the soluble ELP control only exhibited a 4 hr half-life(Betre et al.,
2006). A similar osteoarthritis-based study utilized ELPs fused to the interleukin-1
receptor antagonist (IL-1Ra) to recapitulate bioactivities of the parent molecule(Shamji
et al., 2007).
In the field of neural inflammation, one strategy employed to treat sciatica involved
covalent conjugation of curcumin, a small molecule drug that binds tumor necrosis
59
factor (TNFα). The appended ELP possessed a degradable carbamate linker to mediate
drug release. Studies in vitro confirmed protection against cytotoxicity induced by TNFα
at rates comparable to free drug. Intramuscular injections of curcumin-ELP at the sciatic
nerve, meanwhile, demonstrated 5-fold higher levels of the drug at 96 hrs over its free
counterpart(Sinclair et al., 2013).
Muscle proliferation and differentiation have likewise been investigated in tandem with
ELP protein polymers. H9c2 rat myoblasts were affixed to polystyrene substrates with
two different ELPs either containing or lacking cross-linking domains, and quantitative
fluorescence confirmed greater adhesion and proliferation by these groups relative to a
bare polystyrene control. Measuring the sizes of myotubes during growth further
demonstrated that ELPs (length=980 μm, width=200 μm) outclassed their counterparts
(length=280 μm, width=60 μm) as potential substrates for muscle tissue
regeneration(Ciofani et al., 2013).
1.4.7. Infectious diseases
Anti-infective drugs, such as antibiotics and antivirals, are anticipated to comprise a
market surpassing 80 billion USD by 2017(Informatics.). Since current treatments for
systemic bacterial infections have largely been confined to intravenous antibiotic
delivery, the sustained release potential of injectable, in situ forming ELP depots was
explored (Adams et al., 2009). In this study, ELPs with periodic lysine residues were
covalently cross-linked to β-[Tris(hydroxymethyl) phosphine] proprionic acid (THPP) and
loaded with either vancomycin or cefazolin. Therapeutic concentrations of the two drugs
60
were released at all times. Vancomycin (drug release time constant (τ) =1170 ± 90 hrs)
proved more effective than cefazolin (τ =32 ± 2 hrs) in prolonging release rates at ELP
concentrations of 225 mg/mL. Additionally, both vancomycin and cefazolin ELP depots
exhibited inhibition of B. subtilis. Another small molecule antibiotic, doxycycline, was
explored by Amruthwar and Janorkar,
which focused on delivery from a hydrogel
scaffold(Amruthwar and Janorkar, 2012).
Interestingly, while E. coli remains the
most common organism employed for ELP
biosynthesis, plant-based expression
systems are also gaining traction(Conley
et al., 2009; Floss et al., 2010b). A study
by Scheller and colleagues(Conrad et al.,
2011) used transgenic tobacco plants
(Nicotiana tabacum) to produce ELP
fusions of camel-derived heavy chain
antibodies targeting tumor necrosis factor
(TNF). Previous data from this group
established the viability of this approach
since ELP fusions yielded a 40-fold
increase in scFv levels over conventional
methods(Scheller et al., 2006). In addition,
Figure 9. Serum half-life of
Nt
TNF-
V
H
H
ELP
and neutralization of LPS/D-
Gal toxicity by
Nt
TNF-V
H
H
ELP
. (A)
Three mice were intravenously injected
with 100 μg plant-derived
Nt
TNF-
VHHELP and for control with
100 μg
Ec
TNF-VHH. Serum samples
were prepared over a time range post-
injection. (B) LPS/D-gal-induced septic
shock was blocked by
Nt
TNF-
VHHELP and
Ec
TNF-VHH. Survival was
monitored 24-h post-injection.
Reprinted from [152] with permission of
John Wiley and Sons.
61
the scFv ELPs remained stable within mature seeds for long periods at room
temperature and demonstrated comparable binding affinities to their cognate antigen as
compared to controls lacking an ELP. In this set of studies, the dissociation constants of
anti-TNF ELP (Kd=1.3±0.098 nM) versus its non-ELP, E.coli derived counterpart
(Kd=0.59±0.032 nM) against TNF were comparable. Both constructs likewise prevented
TNF-mediated cell death in vitro in a dose-dependent manner. Strikingly, anti-TNF ELPs
provided an 11.4 hr serum half-life in C57/BL6 mice whereas the non-ELP bound
antibody only exhibited a 28 min half-life (Figure 9A). Subsequent experiments
revealed that the ELP fusion was just as effective as the non-ELP antibody in septic
shock models (Figure 9B).
With regard to antiviral strategies, plant-derived scFv ELPs have been similarly
instructive. As a proof of concept, the 2F5 mAb directed against HIV-1 glycoprotein 41
was adapted into an ELP fusion protein(Floss et al., 2008). The binding kinetics of the
2F5-derived antibodies were similar to CHO-derived controls, but ELPs strangely
yielded absolute antigen binding activities surpassing 100% (152.3% 2F5-ELP versus
94.1%
CHO
2F5), though confounding factors were not elucidated. In follow-up studies,
another anti-HIV mAb (2G12) with neutralizing abilities was also investigated(Floss et
al., 2009). Fusion of ELP to various antibody domains led to increasing declines in
2G12 neutralization activity (IC50= 5.24 to 29.44 μg/mL) relative to the CHO-derived Ab
(IC50=3.71 μg/mL).
62
Certain studies have also employed the ELP platform in the development of novel
vaccine candidates. Seeking a vaccine against the parasitic flatworm Schistosoma
japonicum, Solomon and colleagues(Solomon et al., 2004) designed a cleavable ELP-
thioredoxin linker fused to the 67 kDa Sj67 surface membrane protein. Although the
ELP fusion enhanced solubility of recombinant Sj67, the protein was not amenable to
transition cycling and other methods (e.g. size-exclusion chromatography) were
employed for purification. The preferential IgG binding to rSj67 (4.7-fold greater median
fluorescence) in the sera of 13 schistosomiasis japonica infected volunteers relative to
five uninfected controls was confirmed using bead-based ELISA. Additional diseases for
which potential ELP-based vaccines are being explored include tuberculosis (Floss et
al., 2010a; García-Arévalo et al., 2013), influenza(Ingrole et al., 2014; Phan et al.,
2014), and Psittacine beak and feather disease(Duvenage et al., 2013).
The development of cytotoxic T-lymphocyte vaccine carriers using the previously
mentioned iTEPs has also been described(Cho et al., 2015). In this manuscript, a
diblock copolymer consisting of hydrophobic iTEPA and hydrophilic iTEPB was fused to
pOVA containing the SIINFEKL epitope. The resulting construct formed a micelle with
mean diameters of 81.2 ± 14.2 nm at 5 µM and 71.9 ± 20.8 nm at 25 µM respectively.
After incubation with dendritic cells, surface presentation of SIINFEKL was more
pronounced for the iTEP nanoparticles compared to soluble iTEPB–pOVA or OVA.
Follow-up studies deduced that CD8+ T-cells co-cultured with iTEP-pretreated dendritic
cells exhibited more activity than DC-treated T-cells (4.38-fold), OVA/DC-treated T-cells
(3.81-fold), and iTEPB–pOVA/DC-treated T-cells (2.9-fold). Subcutaneous immunization
63
of C57BL/6 mice likewise revealed that iTEP NPs were capable of activating greater
numbers of cytotoxic T-lymphocytes in vivo than either OVA or free SIINFEKL peptide.
1.4.8 Gene delivery
Since its first exploration in the 1990s, gene therapy—the paradigm of treating diseases
through delivery of corrective genes to the patient—has presented researchers both
with a compelling tool to revolutionize medicine and the unfortunate reality that more
must be done to perfect the technology. Viruses are considered the standard vehicles
for gene delivery where many strategies focus on encapsulating functional genes into a
viral vector capable of transducing target cells. However, this method alone possesses
various pitfalls; gene incorporation via adenoviral vectors is transient due to episomal
regulation(Järås et al., 2007), while retrovirally-introduced genes are prone to random
integration which leads to insertional mutagenesis(Uren et al., 2005).
Adeno-associated virus (AAV), by contrast, is a single-stranded DNA virus that has
been demonstrated to be a safe vector for constitutive, therapeutic expression of
various diseases; another beneficial attribute of AAV lies in its capacity to transduce
both dividing and non-dividing cells for gene delivery(Goncalves et al., 2001; Xie et al.,
2002). Seeking to enhance transduction into human neural stem cells and fibroblasts,
an AAV r3.45 vector generated through directed evolution was adsorbed nonspecifically
onto tissue culture plates containing hydrophobic monoblock ELPs with 128 repeats of
VPGVG(Kim et al., 2012). 24 hr incubation with ELP substrate provided an AAV
immobilization rate greater than 50%. Dissociation kinetics for adsorbed AAV was
64
favorable with <14% of the immobilized vector being released after 4 days to allow for
sustained delivery. As such, the ELP-functionalized AAV system lessened the amount
of vector required to induce cellular transduction when compared against a bolus
delivery control.
Beyond viral gene therapy, ELPs have also been reported to mediate plasmid DNA
delivery(Dash et al., 2011). One study focused on the treatment of critical limb ischemia
(Dash et al., 2015) using an injectable system comprised of ELP hollow spheres within
an in situ-forming ELP gel scaffold to deliver plasmid eNOS and IL-10. H&E stains of
C57BL/6 mouse sections were performed following an in vivo subcutaneous study to
determine blood vessel density (e.g. angiogenesis) and inflammation post-
administration. Two treatment groups, eNOS (20 μg) and IL-10 (10 μg)/eNOS (20 μg),
exhibited higher blood vessel density by day 14 than controls or IL-10 treatment groups.
Inflammation was measured using the volume fraction of infiltrated inflammatory cells.
By day 7, this volume fraction decreased 30% for IL-10 and IL-10/eNOS treatment
groups compared to ELP and eNOS, although no statistically significant difference could
be observed by day 14. Unilateral hind limb ischemia was then induced in the mice to
deduce how treatment groups influence angiogenesis. Assessing clinical severity, the
saline group demonstrated minimal functional recovery with 3/7 animals in the group
exhibiting severely necrotic limbs; the ELP, IL-10, and eNOS groups fared slightly better
than saline. The eNOS/IL-10 group, by contrast, outperformed with 5/7 ischemic mice
experiencing functional recovery. Follow-up studies involving skeletal muscle samples
stained with H&E to further investigate angiogenesis revealed that eNOS and IL-
65
10/eNOS groups were superior to saline, ELP, and IL-10 groups by day 21. In terms of
inflammation, the volume fraction of inflammatory cells was reduced by 50% for IL-10
(10 μg), IL-10 (10 μg)/eNOS (20 μg), and eNOS on day 21 relative to saline.
The rise of gene silencing via RNA interference further equips researchers with another
tool needed to circumvent ailments at the genetic level. First discovered in plants, RNA
interference relies on the cleavage of either endogenous or exogenous dsRNA (e.g.
viral infection, laboratory transfection) by the endoribonuclease Dicer(Hammond, 2005).
This results in the emergence of 21-30 long nucleotides, known as small interfering
RNA (siRNA), which are partitioned into RNA-induced silencing complexes. After
activation, siRNAs acquire the ability to seek and destroy mRNA transcripts in order to
halt protein expression. PeptiMed Inc, for example, has formulated ELPs as part of their
anti-cancer strategy aimed at enhancing uptake and delivery of siRNA targeting the
proto-oncogene EVI1(Primiano, 2015).
1.4.9. Respiratory diseases
Cystic fibrosis, an autosomal recessive disorder arising from defects in the Cystic
Fibrosis Transmembrane Conductance Regulator (CFTR), is the second most common
inherited disease after sickle cell anemia in the US(O'Sullivan and Freedman). This
illness disproportionately affects Caucasians (1 in 2,500 birth incidence) with at least
30,000 Americans suffering from the disease(Zeitlin, 2007). Cystic fibrosis is primarily
characterized by the presence of thick, sticky mucus responsible for blocking the
airways, causing lung damage, and promoting chronic lung infections. Recent studies
66
based at PhaseBio Inc have demonstrated that VIP-ELPs can be repurposed towards
treatment of Cystic fibrosis(Pharmaceutics, 2015). Similar to recent drugs on the market
acting as channel potentiators (e.g. ivacaftor), VIP-ELPs were capable of regulating Clˉ
ions. Most excitingly, VIP-ELP effectively recapitulated both membrane insertion and
channel functioning of misfolded ΔF508 (the most prevalent CFTR mutation) in human
nasal epithelial cells.
1.5 ELPs as Colchicine-Based Nanocarriers
Breast cancers exemplify one common malignancy whose patients would benefit from
this expanded pharmaceutical armamentarium. They remain both the most widely
diagnosed cancer and cause of cancer-related mortality among women with an
estimated 2.1 million cases (~25% of all cancers in women) and over 600,000 deaths
reported worldwide in 2018(Bray et al., 2018); over 268,000 new cases are expected for
both genders within the United States as of 2018(Siegel et al., 2018). Despite
accelerating breast cancer incidence rates across the globe, therapies focused on
specific molecular pathways have significantly reduced mortality rates in high-income
countries. The most successful treatments act on three biomarkers — estrogen
receptor, progesterone receptor, and human epidermal growth factor receptor 2. A
distinct group of tumors known as triple-negative breast cancers (TNBCs), however,
lack these targets and represent an aggressive subtype marked by poor disease
prognosis, high recurrence rates, and diminished overall survival(Hudis and Gianni,
2011). Accounting for 15-20% of all breast cancers, TNBCs are diagnosed more often
among younger women (<40 years old) and constitute the leading cause of breast
67
cancer death in African-Americans(Dietze et al., 2015). As the molecular heterogeneity
of TNBCs have stymied efforts to develop targeted therapies, cytotoxic chemotherapy
provides the only systemic modality despite its attendant drawbacks.
Colchicine, a natural tropolone derivative extracted from Colchicum autumnale and
Gloriosa superba, is approved by the FDA as an anti-inflammatory agent in the
treatment of gout and familial Mediterranean fever(Slobodnick et al., 2018). This drug
was initially investigated for anti-cancer applications owing to its high potency, but a
poor therapeutic index resulting from toxicity to normal cells precludes the use of
colchicine in chemotherapy regimens(Lu et al., 2012). Colchicine, however, continues to
be investigated as a promising pharmacophore for the design of semisynthetic
analogues that can not only enhance efficacy but also reduce toxicity(Sivakumar, 2013).
As microtubule destabilizers, the entire class of colchicinoids act by binding at an
interfacial region of the tubulin heterodimer known as the colchicine binding site; most
interactions with the small molecule occur on the β-subunit thus allowing steric
hindrance of the α-subunit to prevent subsequent microtubule
polymerization(Bhattacharyya et al., 2008).
Elastin-like polypeptides (ELPs) are particularly suited to addressing the hitherto
intractable problems of colchicinoid-based drug delivery given their various applications
as a platform technology. Derived from human tropoelastin, these thermoresponsive
biomaterials can be engineered at the genetic level and typically consist of an iterative
pentameric sequence, [Val-Pro-Gly-Xaa-Gly]n, where Xaa can be any amino acid as the
68
guest residue and n specifies the number of repeats. Coupled with their low
immunogenicity and proteolytic biodegradation, recombinant biosynthesis further allows
precise control over ELP architecture and molecular weight, resulting in monodisperse
nanoparticles suited to the expression of biological moieties as fusion proteins without
sacrificing the functional activity of the parent macromolecule. With respect to drug
delivery, our team has previously established the feasibility of this approach by
exploiting high affinity binding between a small molecule (rapamycin) and its cognate
receptor (FK506 binding protein 12) to design novel drug nanocarriers (FKBP-ELPs) in
models of TNBC and Sjögren’s syndrome(Dhandhukia et al., 2017; Shah et al., 2013b;
Shi et al., 2013). Another study employing a delivery strategy whereby paclitaxel was
chemically conjugated to an ELP, meanwhile, revealed that the resulting ~60 nm
nanoparticles outperformed both free paclitaxel and Abraxane in an orthotopic MDA-
MB-231 mouse model(Bhattacharyya et al., 2015). As such, we hypothesize that two βI-
tubulin ELP nanoformulations with differing solubility profile (TβSI as a coacervate-
forming colchicine depot and TβS96 as a soluble colchicine carrier across
physiologically relevant temperatures) will similarly prove capable of acting as cognate
receptors by binding colchicinoids for anti-tumor efficacy while concomitantly reducing
off-target toxicities against TNBC.
69
1.6 ELPs as Tubulin Purification Tools
One strategy pertinent to streamlining soluble expression from bacterial hosts involves
the design of recombinant proteins linked to solubilizing tags, and as previously
reported, protein polymers serve as exemplary self-purifying tools when genetically
conjugated to parent proteins. Though soluble below transition temperature (Tt), ELPs
exhibit a pronounced yet reversible phase separation above Tt into insoluble
coacervates due to their thermoresponsive nature; this property can be specifically
tailored based on factors such as guest residue identity, molecular weight, and
concentration(Despanie et al., 2016). Protein-ELP fusions likewise acquire
thermosensitivity while retaining their bioactivity, effectively allowing the separation of
fusion proteins from contaminants by leveraging these alternating solubility states.
Known as inverse transition cycling (ITC), this process serves as a non-
chromatographic means of protein purification not unlike existing protocols described in
the literature for temperature-dependent tubulin polymerization / depolymerization
cycling. The methodology outlined for the heterologous generation of human αI/βI
tubulins provides a compelling rationale for designing tubulin-based fusion proteins that
can simplify the current workflow of laboratories relying on bacterial expression systems
70
for tubulin production(Mane et al., 2013). As such, having confirmed the feasibility of
ELPs as solubilizing tags via expression of βI-tubulin fusions, the MacKay laboratory
sought to build on this finding by engineering an αI-tubulin ELP as a heterodimeric
complement to examine the hypothesis that recombinantly generated αβ-tubulin ELPs
can engage in polymerization kinetics akin to native αβ-tubulin (Figure 10). We
therefore detail the expression of a soluble αI-tubulin ELP possessing serine as its
guest residue (TαS96), explore its biophysical properties relative to a prior expressed
βI-tubulin counterpart (TβS96), and subsequently investigate the biological activity of
the resulting nanostructures.
1.7 ELPs as Intracellular Switches
Synthetic biology promises to revolutionize the biomedical field by applying engineering
principles to rationally design biological components which can alter cellular functions in
Figure 10. Overview of α and β tubulins genetically modified with ELPs as
potentially polymerization competent proteins. Tubulin-ELPs consist of the
parent protein fused at the C-terminus to S96, a hydrophilic protein tag that can
facilitate recombinant expression and purification of tubulins from E. coli.
71
a reproducible manner. Tubulin-ELPs are suited as multifunctional intracellular tools to
elucidate microtubule dynamics in cellulo owing to the precise control that can be
exerted over their behaviors within the cytosol. The MacKay group was the first to report
this phenomena in the cytosol — namely, a method of reversibly modifying intracellular
activity using ELP fusion proteins — by exploiting their intrinsic thermo-
responsiveness(Pastuszka et al., 2012). Alterations in temperature as small as 1°C
above a set transition temperature (Tt) promote rapid self-assembly into ELP
microdomains (visualized as puncta) through a process known as inverse thermal
cycling. This process can be tuned to occur across a broad range of physiological
temperatures; below Tt, assembly is effectively reversed thus granting an exquisite level
of control over intracellular functions akin to an on/off switch, hence the term
‘intracellular switching’. Through the reversible manipulation of microdomain formation
in an intracellular milieu, our laboratory previously demonstrated that ELPs can function
as a rapid protein switching system to control clathrin-mediated endocytosis in response
to temperature by sequestering clathrin heavy chain, effectively demonstrating that
intracellular assembly of ELP microdomains can be accomplished in minutes(Pastuszka
et al., 2014). Another promising finding was the fact that ELP microdomains can
undergo either additive coassembly or spatial self-sorting based on their respective
structural properties (e.g. monoblocks versus micellar diblock copolymers). Given its
self-assembly properties, heat-inducible nature, and reversibility, this platform
technology can be specifically harnessed to generate tubulin-ELP fusions capable of
modulating intracellular microtubule dynamics and mitotic arrest. As such, our
hypothesis is that the thermally responsive TβV72 fusion will integrate into the existing
72
pool of cytosolic tubulins and facilitate rapid manipulation of microtubule dynamics in
situ while more generally expanding potential ELP applications throughout the cell.
73
Chapter 2
Materials and Methods
2.1 Materials
All reagents were purchased from Sigma-Aldrich. GST tagged human β-tubulin
(SRP5148) was purchased from Sigma-Aldrich, lyophilized bovine tubulin
(Cat. #142001) was purchased from PurSolutions LLC (Nashville, TN), BLR (DE3) E.
coli competent cells were purchased from EMD Millipore (Billerica, MA), and SHuffle T7
Express Competent E. coli cells (C3029J) were purchased from New England Biolabs
(Ipswich, MA). The genes for human αI- and βI-tubulin were purchased from Integrated
DNA Technologies Inc. The human beta Tubulin (TUBB) C-Myc tag pCMV vector (Cat.
#HG11626-CM) was purchased from SinoBiological (Beijing, China).
2.2 Methods
2.2.1 Gene Design and Synthesis of Bacterially Expressed Tubulin-ELPs
In order to generate the ELPs evaluated, synthetic genes encoding ELP mono and
diblock copolymer fusions with tubulins were constructed. Gene assembly of TβSI as a
diblock copolymer alongside TαS96 and TβS96 as monoblocks was conducted
according to a two-step cloning procedure. The protein sequences were obtained from
UniProtKB with accession number Q71U36 (gene name TUBA1A) for human αI-tubulin
and accession number P07437 (gene name TUBB) for human βI-tubulin. All protein
sequences were converted into DNA sequences and codon optimized for Escherichia
74
coli biosynthesis in EditSeq (DNAStar Lasergene). Two pIDTsmart vectors encoding
TUBA1A and TUBB respectively were ordered from Integrated DNA Technologies (IDT)
containing three restriction cut sites: NdeI, BseRI, and BamHI. In all cases, the
corresponding tubulin gene was directly flanked by the NdeI and BseRI restriction sites
with NdeI and BamHI serving as the 5’ and 3’ termini. TUBA1A and TUBB were each
subcloned from their pIDTsmart vectors via NdeI and BamHI double digestion, gel
purified (Qiagen), and ligated with pet25b (+) vectors also digested by NdeI and BamHI.
The second step entailed insertion of the respective tubulin-pET genes into another
pET25b (+) vector containing the ELP gene sequence via double digestion with BseRI
and BssHII. The correct sequence, insertion, and orientation of the two tubulin-ELP
fusion constructs with TUBA1A and TUBB respectively at the N-terminus followed in-
frame by the ELP were verified via DNA sequencing. Genes coding for ELPs (S96, SI)
were synthesized by recursive directional ligation in pET25b (+) as previously
reported(McDaniel et al., 2010a).
2.2.2 Western Blot Analysis of Tubulin-ELP Inclusion Bodies
8 μg protein from different batches of TβSI containing either initial cold spin
supernatants or bacterial cell pellets were solubilized in Buffer C (Buffer C: 50 mM Tris,
50 mM NaCl, 1 mM CaCl2, 8 M Urea, 10 mM beta-mercaptoethanol, pH 8.8) according
to the methods outlined (Mane et al., 2013) loaded onto SDS-PAGE gels as further
detailed in Determination of Protein Purities below, and protein bands were transferred
by iBlot 2 Dry Blotting System (Thermo Fischer Scientific) onto a nitrocellulose
membrane (IB23002, Thermo Fischer Scientific). The membrane was then blocked with
75
5% w/v nonfat dry milk in Tris buffered saline (TBS) supplemented with 0.1% v/v Tween
20 (TBST - 200 mM Tris, 1.5 M NaCl, pH 7.6) followed by immunoblotting with 10 μL of
mouse primary monoclonal antibody against beta tubulin (Cat. #MA5-11732, Thermo
Fischer Scientific) for a 1:1000 dilution. Following primary immunoblotting, the
nitrocellulose membrane was washed with TBST (3x for 10 min each) and incubated
with 1 μL of secondary horseradish peroxidase (HRP)-linked antibody (Cat. #7074, Cell
Signaling Technology) for 60 min at room temperature to reach a 1:5000 dilution,
washed with TBST (3x for 10 min each), and incubated with HRP substrate (Cat.
#E2400, Denville Scientific, Holliston, MA) for 1 min prior to imaging using
chemiluminescence (ChemiDoc
TM
Touch Imaging System, Bio-Rad).
2.2.3 Expression and Purification of Soluble Tubulin-ELPs
pET25b (+) vectors containing the respective TUBA1A-S96, TUBB-S96, and TUBB-SI
genes were transformed into SHuffle T7 Express Competent E. coli cells prior to
inoculation onto agar plates with 100 µg/mL carbenicillin. After incubation over 18-20
hrs at 30°C for optimal colony growth, a 50 mL terrific broth starter culture
supplemented with 100 µg/mL carbenicillin was inoculated overnight at 37°C in a
shaker. The bacterial culture was inoculated the following day into 3-4 liters of terrific
broth supplemented with 100 µg/mL carbenicillin and grown at 37°C until an OD 600=0.8
was reached. The cells were then induced with 1 mM isopropyl β-D-1-
thiogalactopyranoside for 18 hrs at 25°C. Following IPTG induction, cultures were
harvested by centrifugation at 4,000 rpm for 12 min at room temperature, and cell
pellets obtained from 1 L cultures were each resuspended in 25 mL of Tubulin Buffer A
76
(Tubulin Buffer A: 50 mM Trizma Base, 50 mM MgSO4, 50 mM NaCl, pH 8.8).
Resuspended cells were lysed via Misonix microtip probe sonication with 10 sec on / 20
sec off pulse intervals for 3 min on ice. Polyethyleneimine (0.5%) was added to the
supernatant for co-precipitation of DNA, incubated on ice for 15 min with occasional
agitation, and centrifuged at 4,000 rpm for 12 min at 4°C to discard any insoluble
cellular debris. The supernatant, containing the respective tubulin-ELP fusion proteins,
was purified by 3 rounds of Inverse Thermal Cycling (ITC). Overnight dialysis was
conducted using 10 kDa MWCO SnakeSkin Dialysis Tubing (ThermoScientific) to buffer
exchange the tubulin-ELPs at 4°C against 2 liters of Tubulin Buffer B (Tubulin Buffer B:
25 mM Trizma Base, 25 mM NaCl, 10 mM MgSO4, 1 mM MgCl2, 1 mM CaCl2, pH 7.3).
Purified proteins were filtered through 0.2 µm sterile Acrodisc filters (PN 4612, Pall
Corporation, Port Washington, NY). Protein concentration was spectrophotometrically
deduced by denaturing a 30 µL aliquot of tubulin-ELP stock solution at a 1:1 ratio with
Denaturation Buffer (6 M guanidine hydrochloride, 0.02 M sodium phosphate, pH 6.5) to
calculate ε280 using the Edelhoch method(Pace et al., 1995):
ε280 = (5,500 × # Trp) + (1,490 × # Tyr) + (125 × # Cys) (Eq. 1)
where ε280 represents the calculated molar extinction coefficient according to the Beer-
Lambert law:
Abs = εlc (Eq. 2)
which can be rearranged such that:
M = [(A280 – A350) × dilution factor] / (ε × l) (Eq. 3)
where M specifics molar concentration, A280 and A350 are the absorbances at 280 nm
and 350 nm respectively, l is the path length (cm), and ε is the calculated molar
77
extinction coefficient at 280 nm: 51,810 M
-1
cm
-1
for TαS96 and 48,330 M
-1
cm
-1
for
TβS96 and TβSI respectively. Final yields obtained were 15-30 mg/L.
2.2.4 Determination of Protein Purities and Transition Temperatures
The purity of the fusion proteins was determined using SDS-PAGE. Briefly, 1 to 12 μg of
protein was added to SDS Laemmli loading buffer (1610747, Bio-Rad, Hercules, CA)
containing 10% v/v β-mercaptoethanol then boiled at 95°C for 5 mins prior to being
electrophoresed on a 4-20% precast gradient Tris-Glycine-SDS PAGE gel (58505, Lonza,
Walkersville, MD). Afterwards, the gel was stained using 10% w/v copper chloride and
imaged via ChemiDoc
TM
Touch Imaging system (Bio-Rad); the copper chloride-stained
gel was then rinsed with ddH2O prior to the addition of 50 mL Coomassie Brilliant Blue
R-250 solution (Biorad). The peak areas per gel lane were calculated based on intensity
profiles using ImageJ (National Institutes of Health, Bethesda, MD), and protein purity
was estimated using the following equation:
% purity = (𝐴𝑝𝑒𝑎𝑘 /𝐴 𝑡𝑜𝑡 ) × 100 (Eq. 4)
where Apeak is the area of the peak, and Atot is the total area under all the peaks per
lane.
Transition temperatures were obtained by using optical density measurements at 350
nm in PBS for plain ELPs and Tubulin Buffer B for tubulin-ELPs on a DU 800 UV-Vis
spectrophotometer (Beckman Coulter, Brea, CA). Briefly, increasing concentrations of
constructs were added to 300 μL Tm microcells (Brea, CA), and the temperature was
ramped at a rate of 1°C/min measured every 0.3°C. The optical density was plotted as a
78
function of temperature, and the maximum first derivative of this curve was defined as
the transition temperature. Phase diagrams were then created according to the
following relationship:
Tt = m log10[CELP] + b (Eq. 5)
where Tt is the calculated transition temperature, CELP is concentration, m is the slope,
and b is the intercept temperature transition temperature at a reference concentration of
1 μM.
2.2.5 Secondary Structure Determination Using Circular Dichroism
Circular dichroism was conducted to deduce the secondary structures of the ELP
nanoparticles relative to a bovine tubulin control. Proteins were run on a Jasco J-815
spectrometer (Easton, MD) using a quartz cuvette possessing a ∼1 mm path length. All
proteins were buffer exchanged into filtered ddH2O, and the circular dichroism signal in
millidegrees was monitored from 190 to 250 nm. Spectra were then corrected by
subtracting the spectrum of the ddH2O blank from that of the sample. Scanning of the
protein samples was repeated three times (n=3), and the raw spectra depicted
represent the average of these spectra in triplicate. Deconvolution was performed using
BeStSel analysis program(Micsonai et al., 2018) from 190 – 250 nm based on the mean
residual weight, concentration, and path length for each analyzed protein.
79
2.2.6 Light scattering analyses of nanoparticles
Particle sizes were estimated by measuring hydrodynamic radii (Rh) of the evaluated
proteins via dynamic light scattering. In brief, 25 μM samples in PBS for plain ELPs or
Tubulin Buffer A for Tubulin-ELPs were filtered through 200 nm sterile Acrodisc® 13
mm filters (PN 4454, Pall Corporation). 60 μL of each sample was then loaded in
triplicate into a prechilled 384 well black plate and covered with 20 μL mineral oil. The
plate was centrifuged at 15°C to remove air bubbles and Rh was measured using the
Wyatt Dynapro plate reader (Santa Barbara, CA). Reported values are presented as
mean ± SD (n=3).
Tandem size exclusion chromatography and multiangle light scattering (SEC-MALS)
was employed at 25°C to deduce radii of gyration (Rg), absolute molar masses, and
coordination numbers for nanoparticle samples. Briefly, 60 μM of ELP controls or 10 μM
tubulin and tubulin-ELP proteins respectively were injected into a Shodex KW-803 size
exclusion column using either sterile filtered PBS for plain ELPs or sterile filtered
Tubulin Buffer A (Tubulin Buffer A: 50 mM Trizma Base, 50 mM MgSO4, 50 mM NaCl,
pH 8.8) for tubulin-ELPs at the rate of 0.5 mL/min. Column eluents were then analyzed
on a Wyatt Helios light scattering detector (Santa Barbara, CA), and data was fit to a
random coil plot to determine radius of gyration (Rg ) and absolute molar masses.
Particle morphology was interpreted according to the equation:
ρ = Rg / Rh (Eq. 6)
where Rg specifies radius of gyration and Rh represents the hydrodynamic radius. The
coordination number for nanoparticles was determined by dividing the absolute molar
mass by the calculated molecular weight.
80
2.2.7 MALDI-ToF analysis of molecular weights
In order to provide an alternative means of confirming molecular weight, 1 μL of 10 µM
sample was spotted on the MALDI target and allowed to air dry before 1 μL of 3,5-
dimethoxy-4-hydroxycinnamic acid (sinapinic acid, 10 mg/ml in 50% ACN, 0.1% TFA)
was spotted on the MALDI target and similarly allowed to air dry. The sample was then
analyzed by Microflex-LRF mass spectrometer (Bruker Daltonics, Billerica, MA) in
positive ion, linear mode using a 25 kV accelerating voltage. External calibration was
performed using a protein calibration mixture (4 to 6 proteins) on a spot adjacent to the
sample. The raw data was processed in the FlexAnalysis software (version 3.4.7,
Bruker Daltonics).
2.2.8 Drug retention via extended dialysis
A colchicine standard curve ranging from 1.95 - 500 µM was generated according to a
linear equation, y = 23826x + 34820 to extrapolate fluctuating colchicine concentrations
during experimental analysis. 1 mM Colchicine was subsequently mixed for 30 minutes
at room temperature with 130 μM TβS96 to constitute a ~1:1 drug binding ratio, and 115
μM of the Colchicine / TβS96 admixture was dialyzed in Tubulin Buffer E under 1:650
sink conditions at 4°C using 10 kDa MWCO SnakeSkin Dialysis Tubing (#68100,
ThermoScientific, Waltham, MA). Buffer changes were performed 2x on day 1 with 100
μL aliquots collected from the dialysis tubing at fixed time intervals to track colchicine
concentrations at λ = 350 nm using RP-HPLC. The data was then fit via non-linear
regression according to a one-phase exponential decay model with values presented as
mean ± SD with 95% CI (n = 2).
81
2.2.9 Tubulin polymerization
Optical density was utilized to monitor tubulin polymerization and microtubule formation
for tubulin-ELPs relative to a bovine tubulin control at 37°C. The optical density was
measured via DU 800 UV-Vis spectrophotometer (Beckman Coulter, Brea, CA) using
analytical λ: 340 nm for absorbance within thermal mount microcells (Beckman Coulter)
containing samples. 80 mM piperazine-N,N’-bis[2-ethanesulfonic acid] sequisodium salt
(PIPES), 1 mM MgCl2, and 1 mM ethylene glycol-bis(β-amino-ethyl ether) N,N,N’,N’-
tetra-acetic acid (EGTA) at pH 6.9 was mixed with 15% glycerol and 1 mM guanosine
5’-triphosphate (GTP) sodium salt hydrate at room temperature. Afterwards,
polymerization buffer was pipetted into quartz microcell cuvettes and placed on
microcell stages preheated to 37°C. Tubulin-ELP or Bovine Tubulin samples were
diluted to the appropriate concentration and kept at 4°C before being spiked into the
reaction mixture within the preheated microcells to begin the experiment.
2.2.10 Negative transmission electron microscopy of nanoparticles
Negative transmission electron microscopy was used to observe the dominant
morphology for ELP nanoparticles relative to a bovine tubulin control. For the TEM
protocol, 10 μL of 1 μM protein sample was pipetted onto a Ted Pella carbon/formvar
grid (Redding, CA) and placed in a 37°C incubator for 10 min. Afterwards, the sample
was wicked off by filter paper, and 10 μL of 2% uranyl acetate was added to stain the
sample for 10 min in a 37°C incubator. The uranyl acetate was later wicked off by filter
paper and dried prior to being placed into a JEOL JEM-2100 LaB6 microscope (Tokyo,
Japan). All images were captured under 200 kV accelerating voltage.
82
2.2.11 Pronase treatment of tubulin-ELPs
To deduce whether controlled proteolytic digestion of tubulin-ELPs via pronase
treatment can generate free tubulins, 1 mg / mL of frozen pronase was thawed on ice to
retain enzymatic activity; after thawing is complete, 10X serial dilutions were performed
to obtain 10 µg / mL pronase. 50 µL of 20 µM tubulin-ELPs was then mixed with 50 µL
of 10 µg / mL pronase at 30°C such that final concentrations are 10 µM tubulin-ELP and
5 µg / mL pronase during proteolytic cleavage. After proteolytic cleavage occurred for
the specified amount of time, pronase activity was halted by the addition of 1 µL of 100
mM PMSF directly to the reaction mixture. SDS-PAGE analysis was then conducted in
order to observe proteolytic cleavage.
2.2.12 Gene Design of Mammalian Expressed Tubulin-ELP
Gene assembly of TβV72 was conducted by performing PCR on human beta Tubulin
(TUBB) C-Myc tag pCMV vector to amplify the TUBB-myc sequence with an inserted
BstBI restriction digest site (Top strand: 5′- ccaccaagcttggtaccATGAGGGAA-3’; Bottom
strand: 5′- taaTTCGAAGGCCTCCTCTTCGGCCTCCT-3′). The pcDNA6A-EGFR WT
vector [Mien-Chie Hung, The University of Texas] was obtained via Addgene (#42665,
Cambridge, MA) and EGFR was digested using HindIII and BstBI cut sites while the
TUBB-myc PCR product was similarly digested using HindIII and BstBI and inserted into
the digested pcDNA6A vector to generate TUBB-myc-pcDNA. To further engineer the
TUBB-myc-pcDNA vector, it was digested with AgeI and PmeI to insert an
oligonucleotide sequence (Top strand: 5’-ccggTGCAGCGCTATGATGGCGGCCGC-3’)
83
containing a NotI cut site. The modified TUBB-myc was then digested with AgeI and
NotI while V72 in pET25b (+) was digested with MslI and NotI then ligated to form
TUBB-myc-V72. V72 was synthesized using recursive directional ligation in pET25b (+)
plasmid (MilliporeSigma, Burlington, MA) vector as previously described(McDaniel et
al., 2010b). The final opening frames of the fusion proteins were verified via DNA
sequencing (Retrogen, San Diego, CA).
2.2.13 Transfection and visualization of a mammalian tubulin-ELP
HEK293T cells (#CRL-3216, ATCC, Manassas, VA) were maintained in DMEM
(Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS (#35-011-CV,
Corning, NY) in a humidified incubator with 5% CO2 at 37 °C. For the transient
expression, HEK293T cells were transfected with Lipofectamine 3000 (Thermo Fisher
Scientific, Waltham, MA) and TβV72 at 1000 ng/well with 400,000 cells/well. After 72
hrs, transfected cells were fixed with 4% paraformaldehyde, permeabilized with 0.1%
Triton X-100, blocked with 1% BSA in PBS for 1 hr at room temperature, and incubated
with a mouse anti-myc antibody (#2276, Cell Signaling Technology, Danvers, MA) at
1:1000 dilution for 1 hr at 37 °C. Afterwards, an Alexa Fluor 488-linked rabbit anti-mouse
antibody (#A27023, Thermo Fisher Scientific, Waltham, MA) for 1 hr at 37°C prior to
washing then staining cells with DAPI before mounting. Coverslips were mounted on
microscope slides in Fluoromount (Diagnostic Biosystems, Pleasanton, CA) and images
were captured on a Nikon Diaphot 300 epifluorescence microscope with a Nikon CFWN
10x/20 objective lenses, Nikon CFI Plan Fluor 100XS oil eyepiece lenses, Nikon
Immersion Oil (type F) 30cc PFS, and X-cite 120 LED @ 100% + 488 nm filter.
84
Chapter 3
Results
Table 2. Physical properties of ELP protein polymers with and without tubulins.
ELP
Nomenclature
a,b
Amino Acid
Sequence
c
Calculated
Molecular Weight
(Da)
d
Tt
(°C)
TαS96 TUBA1A-G(VPGSG)96Y 86,691.6 43.2
TβS96 TUBB-G(VPGSG)96Y 87,881.8 68.6
TβSI TUBB-
G(VPGSG)48(VPGIG)48Y
89,133.6 17.7
TβV72 TUBB-myc-
G(VPGVG)72Y
80,396.3 ND
S96 G(VPGSG)96Y 38,228.3 56.9
SI G(VPGSG)48(VPGIG)48Y 39,423.1 26.8,
73.7
a
TUBA1A amino acid sequence [UniProtKB accession number Q71U36]:
MRECISIHVGQAGVQIGNACWELYCLEHGIQPDGQMPSDKTIGGGDDSFNTFFSETGA
GKHVPRAVFVDLEPTVIDEVRTGTYRQLFHPEQLITGKEDAANNYARGHYTIGKEIIDLV
LDRIRKLADQCTGLQGFLVFHSFGGGTGSGFTSLLMERLSVDYGKKSKLEFSIYPAPQ
85
VSTAVVEPYNSILTTHTTLEHSDCAFMVDNEAIYDICRRNLDIERPTYTNLNRLIGQIVSSI
TASLRFDGALNVDLTEFQTNLVPYPRIHFPLATYAPVISAEKAYHEQLSVAEITNACFEP
ANQMVKCDPRHGKYMACCLLYRGDVVPKDVNAAIATIKTKRTIQFVDWCPTGFKVGIN
YQPPTVVPGGDLAKVQRAVCMLSNTTAIAEAWARLDHKFDLMYAKRAFVHWYVGEG
MEEGEFSEAREDMAALEKDYEEVGVDSVEGEGEEEGEEY
b
TUBB amino acid sequence [UniProtKB accession number P07437]:
MREIVHIQAGQCGNQIGAKFWEVISDEHGIDPTGTYHGDSDLQLDRISVYYNEATGGK
YVPRAILVDLEPGTMDSVRSGPFGQIFRPDNFVFGQSGAGNNWAKGHYTEGAELVDS
VLDVVRKEAESCDCLQGFQLTHSLGGGTGSGMGTLLISKIREEYPDRIMNTFSVVPSP
KVSDTVVEPYNATLSVHQLVENTDETYCIDNEALYDICFRTLKLTTPTYGDLNHLVSAT
MSGVTTCLRFPGQLNADLRKLAVNMVPFPRLHFFMPGFAPLTSRGSQQYRALTVPEL
TQQVFDAKNMMAACDPRHGRYLTVAAVFRGRMSMKEVDEQMLNVQNKNSSYFVEWI
PNNVKTAVCDIPPRGLKMAVTFIGNSTAIQELFKRISEQFTAMFRRKAFLHWYTGEGMD
EMEFTEAESNMNDLVSEYQQYQDATAEEEEDFGEEAEEEA
c
Calculated molecular weights based on amino acid sequence
d
Transition temperature (Tt) (25 μM in magnesium buffered solution for tubulin-ELPs
and PBS for ELP controls respectively) determined by optical density measurements at
350 nm; the Tt for mammalian expressed TβV72 was not determined (ND) as of the
completion of this dissertation
86
Bacterially expressed tubulin-ELPs can be obtained in the soluble fraction
In seeking to generate βI-tubulin ELPs, the 89 kDa TβSI was constructed as the initial
fusion protein to be tested in the BLR E. coli expression system. Contrary to initial
expectations, however, TβSI could not be isolated within the supernatant during an
initial cold spin aimed at segregating ELP fusions from cellular debris post-sonication.
As prior reports concerning the expression of human tubulins from E. coli describe
renaturation from inclusion bodies as a necessary step(Mane et al., 2013), we sought to
determine via Western blot analysis whether tubulin-ELPs were similarly being
sequestered (Figure 11). 2 µL aliquots from four different batches of TβSI containing
either initial cold spin
supernatants or solubilized
bacterial cell pellets
revealed that TβSI was
trapped within inclusion
bodies found in the cell
pellets. As opposed to the
labor-intensive
renaturation protocols
employed to obtain
functionally active tubulins
from most E. coli strains,
we identified SHuffle T7 Express as a preferable alternative; this bacterial strain is
specifically optimized via DsbC expression and an oxidizing cytoplasmic environment to
Figure 11. Tubulin-ELPs are sequestered into
inclusion bodies when expressed in BLR E. coli
cells. Western blot analysis revealed that multiple
batches of a tubulin-ELP (TβSI) could not be found in
the supernatant (Lanes 1 – 4) but were present in
bacterial pellets (Lanes 5 – 8) with varying expression
levels at the anticipated MW of 89 kDa relative to a 76
kDa GST-tagged human β-tubulin control (Ctrl).
87
facilitate proper folding of proteins containing multiple disulfide bonds such as the
tubulin family(Lobstein et al., 2012).
SHuffle cells proved integral to the soluble expression of TβSI as a putative depot-
forming drug carrier and led to the subsequent design and expression of TβS96 as a
nanoformulation that can remain in a
soluble, non-coacervated state across
physiologically relevant temperatures.
From six liters each of terrific broth
cultured with TβSI or TβS96
respectively, TβSI was obtained at
yields of 18 mg/L in comparison to 30
mg/L for TβS96.
Each lane of the copper-stained SDS-
PAGE gel was loaded at 8 µg protein
and analyzed via gel densitometry to
quantify protein purity levels (Figure 12, Table 3). In the case of Lanes 1 + 2 (TβSI) and
Lanes 4 + 5 (TβS96), the % purities for both respective lanes were averaged and
reported. Overall, these data establish that heterologous expression of tubulin-ELPs
from a bacterial expression system in a readily soluble form is not only feasible but can
also result in material at ≥80% purity that is suitable for biochemical and biophysical
studies.
Figure 12. TβSI and TβS96 exhibit
soluble expression and can be obtained
at high purity levels. Copper-stained
SDS-PAGE loaded with 8 µg protein to
confirm expression of TβSI (Lanes 1 – 2),
SI (Lane 3), TβS96 (Lanes 4 – 5), S96
(Lane 6), and GST-tagged human β-tubulin
(Ctrl).
88
Table 3. Proteins evaluated via SDS-PAGE in Figure 12.
a
GST amino acid sequence:
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYI
DGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLK
VDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKL
VCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSPGIHRD
b
Calculated molecular weights based on amino acid sequence
c
Purity was determined (Eq. 4) via gel densitometry analysis of copper chloride stained
SDS-PAGE using ImageJ
Lane
Protein
a
Amino Acid
Sequences
b
Calculated
Molecular Weight
(Da)
c
Purity
(%)
1 + 2 TβSI TUBB-
G(VPGSG)48(VPGIG)48Y
89,133.6 80.9
3 SI G(VPGSG)48(VPGIG)48Y 39,423.1 93.9
4 + 5 TβS96 TUBB-G(VPGSG)96Y 87,881.8 85.7
6 S96 G(VPGSG)96Y 38,228.3 92.0
7 Human βI-
Tubulin-GST
TUBB-GST 76,640.4 97.8
89
With the prior expression of TβS96 providing an experimental fait accompli, soluble
expression of TαS96 was similarly accomplished using SHuffle cells, and protein yields
of 15 mg/L were reported from four liters of terrific broth culture. SDS-PAGE was
performed followed by Coomassie staining to confirm the molecular weight of TαS96 in
comparison to TβS96, S96, and Bovine Tubulin (Figure 13A). Given the difficulties in
visualizing the S96 band, this protein had to be loaded at 10 µg relative to the 1 µg
employed for all other proteins; nevertheless, the increased loading amount of S96 did
not enhance its visualization. Differential staining with cupric chloride ameliorated the
obstacle of visualizing S96 thereby permitting gel densitometry analysis to further
Figure 13. Differential staining reveals soluble expression of TαS96 and
TβS96 at high yields. All proteins except S96 were loaded at 1 µg; S96 was
loaded at 10 µg. (A) Coomassie staining confirmed expression of TαS96 (Lane 1),
TβS96 (Lane 2), S96 (Lane 3), and bovine tubulin (Lane 4). (B) Cupric chloride
staining facilitated visualization of 10 µg S96.
A B
A
90
quantify protein purity levels (Figure 13B, Table 4). Akin to prior investigations involving
TβSI and TβS96, this study confirms that both S96-derived tubulin fusions can be
obtained at 85% purity following inverse thermal cycling.
Table 4. Proteins evaluated via SDS-PAGE in Figure 13.
a
Bovine Tubulin amino acid sequence not determined (ND) due to bovine brain tubulin
being comprised of mixed α/β-tubulin isotypes
b
Calculated molecular weights based on amino acid sequence; bovine tubulin estimate
based on monomer, but exact MW determination is impossible due to mixed
composition of α/β-tubulin isotypes
c
Purity was determined (Eq. 4) via gel densitometry analysis of copper chloride stained
SDS-PAGE (Fig. 13B) using ImageJ
Lane
Protein
a
Amino Acid
Sequences
b
Calculated
Molecular Weight
(Da)
c
Purity
(%)
1 TαS96 TUBA1A-G(VPGSG)96Y 86,692 85.7
2 TβS96 TUBB-G(VPGSG)96Y 87,882 85.4
3 S96 G(VPGSG)96Y 38,228 92.8
4 Bovine
Tubulin
ND 55,000 100
91
Tubulin fusions gain ELP-based thermoresponsive properties
As shown in representative UV-Vis optical density profiles and calculated via the first
derivative method, TβS96 exhibits a Tt of 68.6°C at 25 μM, which is in excess of
physiologically relevant temperatures as anticipated, whereas TβSI possesses a Tt of
17.7°C at 25 μM that is below room temperature (Figure 14A). This likely derives from
the fact that - i) the isoleucine block of TβSI mediates hydrophobic interactions; and ii)
Figure 14. TβSI and TβS96 gain ELP-mediated thermoresponsive
behaviors. Representative UV-Vis optical density profiles were obtained by
heating (A) 25 μM Tubulin-ELPs in magnesium buffered solution and (B) 25 μM
ELP controls in PBS respectively at a rate of 0.3°C/min. (C) Optical density data
were collated into temperature-concentration phase diagrams. Dashed lines
indicate the fit of Tt to the following equation: Tt = mlog10[CELP] + b where CELP is
concentration, m specifies the slope, and b represents the transition temperature
at 1 μM.
A B
C
92
the increased molecular weight of βI-tubulin depresses this fusion protein’s transition
temperature thus facilitating coacervation in line with the inverse relationship
established between molecular weight and Tt for ELPs more generally. For the sake of
comparison, 25 μM of hydrophilic monoblock S96 phase separates at 56.9°C while 25
μM of amphiphilic SI essentially has two transition temperatures mediated by its diblock
construction. The first Tt at 26.8°C is defined as its critical micelle temperature (CMT)
and represents the temperature beyond which SI undergoes self-assembly into micellar
nanostructures owing to the hydrophobic interactions of the isoleucine block; the second
Tt of 73.7°C, meanwhile, indicates the temperature at which bulk phase separation of SI
is precipitated by the hydrophilic serine block (Figure 14B). In further comparing the two
Tubulin-ELPs, the impact of selecting different ELP backbones (i.e. SI, S96) becomes
especially pronounced since appending hydrophilic S96 to βI-tubulin results in a fusion
protein (TβS96) that surprisingly surpasses the Tt of S96. Although these data may
appear in contravention of the inverse relationship between ELP molecular weight and
transition temperatures prima facie(Despanie et al., 2016), it is likely that the iterative
hydroxyl groups present in TβS96 due to serine serving as its ELP guest residue
enforce a higher heat requirement for this fusion protein before the hydrophobic
interactions characteristic of ELP phase separation is triggered(Urry et al., 1985). TβSI
similarly represents a departure from the typical behavior of its ELP scaffold since it
exhibits only one Tt , compared to the CMT and bulk Tt described for SI, thereby
yielding initial evidence that this fusion protein may exist a priori as pre-assembled
nanostructures. By employing a sample range of concentrations (e.g. 5, 10, 25, 50, and
100 μM), Tt can then be extrapolated for these ELPs at any concentration and
93
visualized using a concentration-temperature phase diagram (Figure 14C) based on the
linear equation, Tt = m log10[CELP] + b, where C ELP is the concentration, m specifics the
slope, and b represents the transition temperature at a reference concentration of 1 μM
(Table 5).
Table 5. Quantification of Temperature-Concentration Phase Diagram in Figure 14C.
a,b
Phase diagrams (Figure 13C) were fit with Eq. 5 (Tt = mlog10[CELP] + b). Values
represent mean ± 95% CI.
Most notably, TβSI was found to display less ELP-mediated concentration dependence
as demonstrated by a markedly diminished slope relative to the SI control. Prior reports
from our laboratory, however, established that certain macromolecular partners (e.g.
Protein
a
Slope, m
[°C Log(μM)]
b
Intercept, b
[°C]
TβSI -0.3 ± 0.4 18.0 ± 0.6
TβS96 -2.5 ± 1.7 71.4 ± 2.4
S96 -3.3 ± 1.9 62.1 ± 2.7
SI
(CMT)
-2.8 ± 3.5 30.0 ± 4.4
SI
(Bulk Tt)
-2.6 ± 2.5 77.0 ± 3.1
94
disintegrin, lacritin) can also attenuate this concentration-dependent relationship once
fused to hydrophobic ELPs as a corollary of the parent protein’s tendency to drive de
novo nanoparticle assembly(Janib et al., 2014a; Wang et al., 2014b); these data thus
complement the earlier supposition that βI-tubulin may serve as a globular core driving
the formation of pre-assembled nanostructures during protein expression.
Representative UV-Vis optical density profiles comparing TαS96 and TβS96 at 25 µM
reveal that, while these tubulin fusions remain stable across a range of temperatures
(10°C - 40°C), TαS96 exhibits a diminished Tt of 43.2°C relative to the TβS96 Tt at
63.8°C (Figure 15A). Interestingly, the 25 µM S96 control’s Tt at 56.3°C is largely
obscured by the higher optical densities observed for tubulin fusions owing to their
existence as pre-assembled nanostructures capable of scattering light. To extrapolate Tt
at any given concentration according to the linear equation, Tt = m log10[CELP] + b,
Figure 15. TαS96 and TβS96 remain stable at physiologically relevant
temperatures similar to S96. (A) Representative UV-Vis optical density profile
was obtained by heating 25 μM Tubulin-ELPs and S96 control in magnesium
buffered solution at a rate of 0.3°C/min. (B) Optical density data were collated into
temperature-concentration phase diagrams. Dashed lines indicate the fit of Tt to
the following equation: Tt = mlog10[CELP] + b where CELP is concentration, m
specifics the slope, and b represents the transition temperature at 1 μM.
A B
95
TαS96 was tested across a sample concentration range (e.g. 3.125, 6.25, 12.5, 25 and
50 μM) and compared to the previously plotted concentration range (e.g. 5, 10, 25, 50,
and 100 µM) reported for TβS96 and S96 respectively (Figure 15B). Relative to TβS96,
TαS96 experiences a more pronounced concentration-dependent decrease in Tt (Table
6). Given that both tubulin fusions share S96 as their ELP backbone, a greater
prevalence of hydrophobic amino acid residues within the αI-tubulin sequence might
account for this Tt disparity between TαS96 and TβS96.
Table 6. Quantification of Temperature-Concentration Phase Diagram in Figure 15B.
a,b
Phase diagrams (Figure 14B) were fit with Eq. 5 (Tt = mlog10[CELP] + b). Values
represent mean ± 95% CI.
Protein
a
Slope, m
[°C Log(μM)]
b
Intercept, b
[°C]
TαS96 -15.3 ± 6.1 67.6 ± 7.2
TβS96 -2.5 ± 1.7 71.4 ± 2.4
S96 -3.3 ± 1.9 62.1 ± 2.7
96
Tubulin fusions retain secondary structures resembling a bovine tubulin control
Circular dichroism spectroscopy, a technique measuring the differential absorption of
left and right circularly polarized light, serves as a useful assay for determining the
secondary structure of polypeptides and proteins in the far-ultraviolet range (190-250
nm) based on protein chromophores derived from peptide bonds, amino acid side
chains, and any associated prosthetic groups; furthermore, CD also provides a
benchmark for comparing the conformational changes observed within the spectra of
engineered fusion proteins relative to spectra from the native parent protein(Greenfield,
2006). The raw CD spectra observed at 25°C represent an average (n=3) of each run
from which bands of characteristic shapes and magnitudes can be identified (Figure
16A). Native bovine brain tubulin was employed as a control, and the resulting far-UV
spectrum comports with prior CD data reported in the tubulin literature (de Pereda et al.,
1996; Lee et al., 1978); the spectrum qualitatively reveals that bovine tubulin is rich in α-
Figure 16. Tubulin fusions retain secondary structures resembling a bovine
tubulin control. All studies were carried out over the far-ultraviolet range (250 nm -
190 nm) to assess secondary structure. (A) Depicted CD spectra represent an
average (n=3) conducted at 25°C. (B) Comparison of 10 μM TβSI CD spectra below
(4°C) and above (25°C) its Tt with 10 μM SI as a control (n=3).
A B
97
helical content as denoted by two negative bands at 221 and 209 nm, respectively,
coupled with a positive band at 191 nm. Interestingly, TαS96 and TβS96 both exhibit
spectra akin to the bovine tubulin control, although the intensities of the negative bands
in the TβS96 spectrum are considerable weaker relative to its positive band at 191 nm.
Interestingly, the intensities of TαS96 are more pronounced relative to its TβS96
counterpart in terms of the positive band at 191 as well as a negative band at 225 nm.
TβSI, by contrast, possesses a spectrum completely at variance with the bovine tubulin
control since its Tt is well below the temperature of 25°C utilized in this experiment;
given that ELPs are reported to overwhelmingly favor type II β-turns following
coacervation, this aligns with the negative band ranging from 220-230 nm for TβSI. For
the sake of comparison, the spectra for S96 and SI were included as soluble ELP
controls with their pronounced negative bands at 200 nm corresponding to a random
coil conformation. Owing to the thermosensitivity of TβSI, raw CD spectra were taken to
compare differences in its secondary structure at 4°C (n=3) relative to 25°C with SI as
an ELP control (Figure 16B). Below its Tt at 4°C, TβSI exhibits a CD spectrum
pronounced in α-helical content akin to native bovine tubulin, unlike its β-turn rich
coacervated state at 25°C, whereas both SI spectra retain the random coil conformation
since these temperatures are below its Tt.
While CD spectra yield a raw description of the data, investigators have also developed
methods for conducting further structural analysis. The most common methodology
assumes that a far-UV CD spectrum represents the linear sum of each contributing
secondary structure (e.g. α-helix, β-sheet) within the protein conformation which can be
98
individually calculated and quantitatively weighted according to its abundance. A caveat
to this approach, however, lies in the fact that ‘pure’ secondary structures do not exist in
nature and thus exemplify a theoretical construct by necessity; in addition, CD spectral
data often fail to accurately predict β-sheet content owing to the broad diversity of these
specific secondary structures. To address these drawback, a web-based tool known as
BeStSel (Beta Structure Selection) has been pioneered to analyze and segregate
experimental CD spectra into eight different secondary structural components based on
the Dictionary of Secondary Structure of Proteins (DSSP)(Micsonai et al., 2018;
Micsonai et al., 2015). Spectra obtained from BeStSel are optimized, according to the
SP175 reference dataset containing CD spectra for 73 proteins corresponding with
high-quality crystal structures in the Protein Circular Dichroism Data Bank, to obtain
model fits for comparison against the deconvoluted spectra produced from these
studies (Figure 17).
99
Having established the general accuracy of deconvoluted spectra, quantitative data
were extracted from BeStSel and assigned secondary structural estimations according
to the following classifications: α-helix, β-sheet, turn, and unordered (Table 7).
B
C
D E
Figure 17. Deconvolution confirms that soluble tubulin fusions exhibit
secondary structures similar to bovine tubulin. (A) TαS96 at 25°C; (B) TβS96
at 25°C; (C) TβSI at 4°C and at 25°C; (D) Bovine Tubulin at 25°C; (E) S96 at 25°C;
(F) SI at 4°C and at 25°C. All raw CD spectra were deconvoluted over the far-
ultraviolet range (250 nm - 190 nm) according to the BeStSel method. Units on y-
axis are defined as molar ellipticity.
A
B
C
E
D
F
100
Table 7. BeStSel estimated secondary structure content (%)
Structural
Motif
TαS9
6
TβS96
TβSI
(4°C)
TβSI
(25°C)
S96
SI
(4°C)
SI
(25°C)
Bovine
Tubulin
α-helix 0.0 0.0 0.0 0.0 0.0 0.0 0.0 15.4
β-sheet 44.2 43.0 43.0 43.8 42.8 42.3 42.6 33.7
Turn 14.6 14.3 14.3 14.4 14.2 14.4 14.6 13.0
Unordered 41.2 42.7 42.7 41.8 42.9 43.3 42.8 37.8
Total 100 100 100 100 99.9 100 100 99.9
According to the BeStSel method, only bovine tubulin exhibited a shift in structural
elements owing to its enhanced proportion of α-helical content (15.4%). The 0.0% total
reported herein for the tubulin fusions may underestimate their true α-helical
breakdown, however, as prior studies investigating tubulin secondary structure via
circular dichroism spectroscopy have reported α-helical contents ranging from 26 to
33%(de Pereda et al., 1996; Lee et al., 1978). This could possibly be attributed to the
fact that, despite providing amenable model fits, BeStSel might remain ill-suited to
accurately parsing secondary structures for artificial proteins featuring iterative motifs
such as the ELP fusion proteins. Indeed, while analytical methods adapted for deriving
data from circular dichroism spectroscopy can prove useful for characterizing secondary
structural changes, this technique nevertheless has limited accuracy in absolute terms.
101
Soluble tubulin fusions derived from a hydrophilic ELP are stable across a broad
temperature range
Dynamic light scattering (DLS), which is based upon the principles of Brownian motion,
was utilized as a means of evaluating nanoparticle size via the hydrodynamic radii of
these fusion proteins at 25 µM
(n=3, mean ± SD) across a range
of temperatures from 10°C to 40°C
(Figure 18A). At 10°C, TβS96
exhibited an Rh = 69.5 ± 0.3 nm in
comparison to TβSI which had an
Rh = 61.4 ± 2.9 nm. 25 µM TβSI,
however, underwent phase
separation beyond 17°C thus
rendering all subsequent DLS
measurements as unreadable due
to multimodal distribution, a finding
which corroborates the previously
conducted UV-Vis temperature
ramps. TβS96, by contrast,
remained soluble at 37°C with an
Rh = 63.8 ± 1.5 nm. Another
experiment was subsequently
Figure 18. TβS96 is a more physiologically
relevant nanoformulation than TβSI. (A)
Hydrodynamic radii (Rh) of βI-tubulin ELPs
reveal that TβSI undergoes coacervation and
precipitation beyond 17°C whereas TβS96
remains soluble at 37°C (n=3, mean ± SD). (B)
Similar to its S96 ELP backbone, TβS96 retains
a stable Rh at 37°C over 72 hrs (n=3, mean ±
SD).
A
B
102
conducted to assess the stability of TβS96 at 37°C over 72 hrs relative to S96 as a
control (Figure 18B). For this population of TβS96 nanoparticles, Rh = 58.6 ± 0.4 nm
after 72 hrs in comparison to the S96 Rh = 4.8 ± 0.0 nm, a finding that seemingly
demonstrates no variance at the measurement resolution investigated. Attempting to
report this measurement with additional significant figures beyond what is afforded by
the instrument, however, would create the illusion of false precision.
Since the S96-based tubulin fusions are known to remain soluble across physiologically
relevant temperatures, dynamic light scattering (n=3, mean ± SD) was instead
employed to measure intensity at 25°C on 25 µM samples of TαS96, TβS96, S96, and
Bovine Tubulin respectively (Figure 19). Regarding the two fusions, TαS96 possessed
a Rh = 79.6 ± 1.5 nm in contrast to TβS96 which exhibited a smaller Rh = 54.5 ± 0.3 nm
for the population of nanoparticles under investigation. In terms of control samples, on
Figure 19. TαS96 exhibits hydrodynamic radii slightly larger than TβS96.
25 µM samples were employed for each sample and all measurements were
conducted at 25°C (n=3, mean ± SD).
103
the other hand, S96 nanoparticles, displayed a Rh = 5.3 ± 0.2 nm while Bovine Tubulin
had a Rh = 63.0 ± 1.2 nm owing to its dimeric makeup. Overall, the hydrodynamic radii
reported for TαS96 and TβS96 not only lend further credence but also provide
additional evidence that these tubulin fusions drive de novo nanoparticle formation
which likely accounts for the intense light scattering they exhibit on optical density
profiles even in their soluble state.
104
Tubulin fusions are not amenable to MALDI-ToF analysis
Figure 20. MALDI-ToF analysis confirms the molecular weight of S96 but
not TβS96. 10 µM of both samples were air dried at 1 μL prior to the addition of
10 mg/mL sinapinic acid (50% ACN, 0.1% TFA) at 1 μL. (A) The major peak
intensity observed for S96 at 38,343 Da aligns with our calculated MW of
38,228 Da. (B) In contrast, the major peak intensity for TβS96 at 40,439 Da
differs markedly from its calculated MW of 87,882 Da.
A
B
105
Figure 21. MALDI-ToF analysis cannot accurately confirm the molecular
weight of TβSI. 10 µM of both samples were air dried at 1 μL prior to the
addition of 10 mg/mL sinapinic acid (50% ACN, 0.1% TFA) at 1 μL. (A) The
smaller peak intensity observed for SI at 39,264 Da comports with our
calculated MW of 39,423 Da. (B) TβSI, on the other hand, possesses multiple
peak intensities that prevent the deduction of an accurate MW relative to its
calculated MW of 89,134 Da.
A
B
106
In order to provide a complementary technique to confirm the calculated molecular
weights conducted via SDS-PAGE for tubulin fusions and their ELP backbone
counterparts to a greater degree of precision, matrix assisted laser desorption ion time
of flight (MALDI-ToF) mass spectrometry was attempted. In the case of S96, the major
peak intensity occurred at a mass-to-charge ratio (m/z), corresponding to an observed
MW= 38,343 Da, which not only comports with the calculated MW of 38,228 Da but also
agrees with previous reports on S96 from our laboratory (Figure 20A). TβS96, on the
other hand, exhibited its greatest peak intensity at a m/z in alignment with an observed
MW= 40,439 Da which conflicts with an anticipated MW = 87,882 Da based on its
amino acid residues (Figure 20B). Interestingly, SI had a m/z correlating to an observed
MW = 39,264 Da which aligned with prior reports even though this peak did not
represent the most significant peak intensity (Figure 21A). A specific peak intensity
corresponding to TβSI, meanwhile, could not be identified owing to the various peaks
found within its spectrum (Figure 21B). Overall, the MALDI-ToF spectra obtained for
S96 confirms that this technique can yield precise molecular weight determinations,
although multiple factors (e.g. specific acidic matrix compound added, solvents applied,
and protein concentration) influence accurate spectral readings(Leurs et al., 2016).
Regarding discrepancies seen in the observed MWs of SI and the β-tubulin fusions, one
potential explanation is that air drying of the matrix solution / protein sample admixture
to facilitate vaporization of volatile solvents at temperatures ≥ Tt may precipitate ELP
coacervation ergo rendering it nigh impossible to ionize and identify intact protein
fractions. While this line of reasoning may suffice to describe the multiple peak
intensities seen in the TβSI spectrum given its established Tt below 25°C, it still cannot
107
account for the higher Tt of TβS96; in this case, an alternative hypothesis supported by
DLS hydrodynamic radii measurements could be that β-tubulin fusions are ill-suited for
MALDI-ToF analysis owing to its signal intensity discrimination against higher mass
oligomers thus MALDI-ToF was not conducted on TαS96. A third possible explanation is
that TβS96 and TβSI may have been cleaved. Given that arginine and lysine residues
typically act as substrates for common proteases(Olsen et al., 2004), Arg-359 and Lys-
362 represent potential sites capable of producing cleavage products (39,654 Da and
39,980 Da respectively) similar to the observed MW= 40,439 Da for TβS96; the five
cleavage products observed for TβSI, meanwhile, render it more difficult to estimate
potential cleavage sites given that the βI-tubulin amino acid sequence possesses 22
arginines and 15 lysine residues.
Tubulin-ELPs assemble into nanoparticles rich in tubulin moieties relative to a
bovine tubulin control
Figure 22. Bovine tubulin exhibits the morphological features of a monomer in
PBS. All measurements were conducted at 25°C and representative chromatograms
were depicted for both proteins. (A) As a monomeric protein control, 30 µM bovine
serum albumin possessed a higher MW at 7.804E4 Da relative to its expected MW of
66.5 kDa. (B) 10 µM bovine tubulin (n=3) exhibited a MW=5.619E4 Da that comports
with the reported MW for tubulin in a dilute, monomeric state.
A
B
108
As an alternative means of calculating the molar masses, in addition to complementing
DLS measurements by further elucidating biophysical properties such as particle
morphology, sample analysis via size exclusion chromatography in line with multi-angle
light scattering (SEC-MALS) was conducted (n=3) at 25°C. Given its expected
molecular weight of ~66,500 Da, Bovine serum albumin (BSA) was selected as a
protein control to establish the accuracy of our SEC-MALS results; interestingly, while
the chromatogram confirmed the presence of BSA monomers, this eluate nevertheless
yielded a molecular weight of ~78,000 Da which is larger than the value reported in the
literature (Figure 22A, Table 8). Bovine Tubulin’s observed molecular weight of 56,190
Da, meanwhile, offers a precise measurement given that researchers typically report a
~55,000 Da molecular weight for tubulin when diluted down to the monomeric
state(Luduena et al., 1977) (Figure 22B).
The ρ factor, defined as the ratio of Rg:Rh, can yield a morphological description of
proteins in solution when paired with values obtained from DLS measurements(Hong et
al., 2012). As molar mass references, the theoretical value for a hard sphere is 0.778
whereas a stiff rod is 2.36; intermediate values for ellipsoids, meanwhile, range from
0.875 to 0.987 for oblate ellipsoids versus 1.36 to 2.24 for prolate ellipsoids.
109
Table 8. Quantification of SEC-MALS Chromatogram in Figure 22.
Protein
a
Observed
Molecular
Weight
(Da)
b
Radius
of
Gyration
(nm)
c
Calculated
Coordination
Number
d
Calculated
Shape
Factor
Expected
Morphology
Bovine
Serum
Albumin
7.804 ±
0.068E4
16.2 ±
1.9
1.2 ND ND
Bovine
Tubulin
5.619 ±
0.077E4
91.2 ±
0.6
1.0 1.4 Prolate
ellipsoid
a,b
Observed molecular weight and radius of gyration determined by size exclusion
chromatography-multiangle light scattering
c
Calculated coordination number determined by dividing observed molecular weight by
calculated molecular weight
d
Calculated shape factor determined according to Eq. 6 (ρ = Rg / Rh) based on previous
dynamic light scattering measurements; not determined (ND) for bovine serum albumin
110
Molar mass distributions from one representative run were depicted for 60 µM SI and 10
µM TβSI respectively (Figure 23). With regard to the two predominant peaks observed
for SI, Molar Mass Fit 1 corresponding to 2.664E8 g/mol and a radius of gyration =
113.7 ± 1.8 nm likely constitutes an oligomeric fraction since general SEC principles
dictate that higher MW fractions elute more rapidly than fractions of lower MW; another
means of interpreting this prodigious molar mass (2.664E8 g/mol) involves dividing it by
SI’s calculated molecular weight (39,423 g/mol) from Table 2 to estimate that each
fraction 1 oligomers consist of approximately 6,758 SI moieties (Figure 23A, Table 9).
The second fraction with its radius of gyration = 53.4 ± 60.5 nm likely also represent an
oligomeric eluate since 7.7 SI moieties could reasonably comprise this fraction.
Figure 23. TβSI possesses three distinct fractions with disparate
morphological features. All measurements were conducted at 25°C (n=3) and
representative chromatograms were depicted for both proteins. (A) 60 µM SI elutes
as two fractions possessing 6758 moieties and 8 moieties respectively in PBS. (B)
10 µM TβSI in tubulin buffer elutes as three fractions with 324 moieties in fraction 1
while fractions 2 and 3 exist as dimers.
A B
111
Table 9. Quantification of SEC-MALS Chromatogram in Figure 23A.
SI
Fraction
a
Observed
Molecular
Weight
(Da)
b
Radius of
Gyration
(nm)
c
Calculated
Coordination
Number of
Nanoparticles
1 2.664 ±
0.131E8
113.7 ± 1.8 6,757.5
2 3.040 ±
0.038E5
86.3 ± 67.3 7.7
a,b
Observed molecular weight and radius of gyration determined by size exclusion
chromatography-multiangle light scattering
c
Calculated coordination number of nanoparticles determined by dividing observed
molecular weight by calculated molecular weight
112
In making this assessment concerning the imprecise SI values obtained, one should
recall that both the nature and number of ELP coacervates observed via SEC-MALS are
subject to a myriad of differing experimental factors (e.g. monomeric molecular weight,
protein concentration, buffer conditions, and temperature); to that end, any data
interpretations gleaned from this SI chromatogram must perforce remain subject to the
caveat that these data might have been affected by ELP self-assembly.
Bearing these analytical caveats in mind, alongside TβSI’s propensity to coacervate
below the 25°C temperature maintained during SEC-MALS analysis, three fractions
were identified from the 10 µM TβSI chromatogram (Figure 23B, Table 10). The
reported radii of gyration values for Fractions 2 and 3 are particularly imprecise, yet they
nevertheless exhibit a similar number of moieties per nanoparticle when divided by the
calculated TβSI MW of 89,133.6 (Table 2) thereby suggesting that these later-eluting
fractions might exist as dimers.
113
Table 10. Quantification of SEC-MALS Chromatogram in Figure 23B.
TβSI
Fraction
a
Observed
Molecular
Weight
(Da)
b
Radius
of
Gyration
(nm)
c
Calculated
Coordination
Number of
Nanoparticles
d
Calculated
Shape
Factor
Expected
Nanoparticle
Morphology
1 2.889 ±
0.102E7
62.6 ± 3.0 324.1 1.0 Oblate
ellipsoid
2 1.613 ±
0.011E5
37.2 ±
73.9
1.8 0.6 Sphere
3 1.586 ±
0.004E5
77.2 ±
52.8
1.8 1.3 Prolate
ellipsoid
a,b
Observed molecular weight and radius of gyration determined by size exclusion
chromatography-multiangle light scattering
c
Calculated coordination number of nanoparticles determined by dividing observed
molecular weight by calculated molecular weight
d
Calculated shape factor determined according to Eq. 6 (ρ = Rg / Rh) based on previous
dynamic light scattering measurements
114
Based on the Rg for each of the three TβSI fractions divided by the prior reported Rh
value of 61.4 nm, shape factors corresponding to various forms were identified. Just as
with DLS, however, TβSI’s low Tt ultimately prevents an accurate derivation of
physicochemical properties via SEC-MALS at physiologically relevant temperatures.
Representative chromatograms detailing molar mass distributions were likewise
depicted for TαS96 and TβS96 at 10 µM while S96 was performed at 60 µM (Figure
24). Each of the molar mass fits corresponding to TαS96, TβS96, and S96 represent
single fractions comporting with polymeric constitutions. S96’s observed molecular
weight of 39,550 Da particularly aligns with previously described values from our
laboratory. By contrast, both TαS96 (1.477E8 Da) and TβS96 (4.858E7 Da) exhibit
molecular weights which are orders of magnitude larger than the control samples and
particularly indicative of their nature as aggregate-like nanoparticles. As such, a more
suitable method involves dividing the respective molecular weights obtained via SEC-
MALS by the prior calculated molecular weights to estimate the number of moieties
Figure 24. TαS96 and TβS96 consist of tubulin-compacted oligomers. All
measurements were conducted at 25°C (n=3) and representative chromatograms
were depicted for all proteins. (A) 10 µM TαS96 in tubulin buffer A elutes as one
fraction possessing 1704 moieties. (B) 10 µM TβS96 in tubulin buffer A elutes as one
fraction possessing 553 moieties. (C) 60 µM S96 in PBS exhibits the morphological
features of a hydrated coil with an observed MW of 39.5 kDa that comports with its
calculated MW of 38.2 kDa.
A
B
C
115
comprising each nanoparticle; TαS96 yields ~1,704 αI-tubulin moieties relative to the
~553 βI-tubulin moieties calculated for TβS96 which could account for TαS96’s
diminished Tt reported in the earlier UV-Vis spectrophotometry data (Figure 15, Table
6). The shape factors calculated for TαS96, TβS96, and Bovine Tubulin correspond to
prolate ellipsoids whereas S96 was considered more akin to a hydrated coil (Table 11).
Table 11. Quantification of SEC-MALS Chromatogram in Figure 24.
Protein
a
Observed
Molecular
Weight
(Da)
b
Radius
of
Gyration
(nm)
c
Calculated
Coordination
Number of
Nanoparticles
d
Calculated
Shape
Factor
Expected
Nanoparticle
Morphology
TαS96 1.477 ±
0.002E8
99.0 ± 0.7 1,703.7 1.2 Prolate
ellipsoid
TβS96 4.858 ±
0.036E7
88.3 ± 0.4 552.8 1.6 Prolate
ellipsoid
S96 3.955 ±
0.0E4
11.4 ± 2.1 1.0 2.2 Hydrated coil
a,b
Observed molecular weight and radius of gyration determined by size exclusion
chromatography-multiangle light scattering
c
Calculated coordination number of nanoparticles determined by dividing observed
molecular weight by calculated molecular weight
116
d
Calculated shape factor determined according to Eq. 6 (ρ = Rg / Rh) based on previous
dynamic light scattering measurements
Tubulin fusions exhibit colchicine-induced quenching
Fluorescence spectroscopy was the final technique conducted to deduce the biological
activity of TβSI, TβS96, and TαβS96 after mixing inside a fluorescence cuvette to yield
a final concentration of 2 µM TαβS96 relative to a 1 µM bovine tubulin control (Figure
25). This assay exploits the quenching of intrinsic tubulin fluorescence induced by
colchicine due to forster resonance energy transfer from excited tubulin tryptophans
colocalized near the colchicine binding site found on β-tubulin to the small molecule
Figure 25. Colchicine absorbs tubulin fluorescence emission in a
concentration-dependent manner. Emission spectra observed at 25°C (n=3) for
intrinsic tubulin tryptophan fluorescence quenching at excitation λ = 295 nm by
holding (A) Bovine Tubulin (B) TαβS96 (C) TβS96 or (D) TβSI constant at 1 µM
while titrating colchicine concentrations (0 – 120 µM).
A
B
C D
117
itself(Bhattacharyya et al., 2010). Despite prior difficulties in deducing its
physicochemical properties, TβSI nevertheless exhibited biological activity in the form of
concentration-dependent quenching
akin to both TβS96 and the bovine
tubulin control. The TαβS96
admixture, meanwhile, possessed
fluorescence intensities far exceeding
those reported for the Bovine Tubulin
control and exhibited fluorescence
quenching in a concentration-
dependent manner comparable to
other samples. Considering the
collated TβSI data in toto, however,
this particular ELP nanoformulation
would be ill-suited as a colchicine
nanocarrier relative to TβS96 owing to
its Tt below room temperature. To that
end, TβS96 was selected as the
appropriately soluble nanoformulation
incapable of coacervation across
physiologically relevant temperatures
for studies investigating βI-tubulin
ELP as a viable colchicine-based
Figure 26. TβS96 does not enhance
colchicine retention. (A) A colchicine
standard curve was plotted based on a
range (1.95 - 500 µM) of drug
concentrations according to a linear
equation, y = 23826x + 34820, with R² =
0.9998. (B) Dialysis followed by RP-HPLC
analysis revealed that TβS96 could not
retain colchicine based on its elimination t1/2
= 1.7 ± 0.4 hrs. Data were fit according to a
one-phase exponential decay model with
values presented as mean ± SD with 95%
CI (n = 2).
A
B
118
nanocarrier.
A soluble tubulin fusion does not enhance colchicine retention in vitro
The next experimental dataset focuses on the viability of the TβS96 nanoformulation as
a soluble drug delivery vehicle capable of ferrying colchicine. A colchicine standard
curve ranging from 1.95 - 500 µM was generated (Figure 26A) to extrapolate fluctuating
colchicine concentrations during experimental analysis. As a means of determining
whether TβS96 enhances colchicine retention, dialysis was then conducted under sink
conditions followed by the collection of 100 µL aliquots to assay colchicine
concentrations via reverse phase high performance liquid chromatography under the
assumption that colchicine would exhibit burst release from TβS96 according to one-
phase exponential decay. Relative to an elimination t1/2 = 1.6 ± 0.3 hrs for the free
colchicine control, however, TβS96 did not discernibly prolong the drug’s release profile
based on its elimination t1/2 = 1.7 ± 0.4 hrs (Figure 26B). The reduction of drug
concentrations within the TβS96 + colchicine study to undetectable levels following an
initial buffer change further disproves our hypothesis that recombinant β-tubulin ELPs
can act as cognate receptor-based nanocarriers by facilitating high affinity binding and
delivery of colchicine.
119
αβ tubulin fusions seemingly self-assemble to recapitulate polymerization
kinetics yet remain unresponsive to microtubule inhibitor ligands
Tubulin polymerization, a quintessential property of tubulin subunits as cytoskeletal
components in cellulo, was assayed to deduce whether recombinant tubulin-ELPs retain
this native activity under in vitro polymerization conditions(Mirigian et al., 2013) relative
to a bovine tubulin control (Figure 27). As anticipated, the 15 µM TαβS96 sample
reproducibly exhibited the three characteristic phases of tubulin polymerization —
namely, nucleation, growth, and steady state equilibrium — although the pronounced
optical density values indicate that TαβS96 nanoparticles scatter light more prolifically
relative to the bovine tubulin control.
Figure 27. TαS96 and TβS96 appear to mimic microtubule dynamics while also
potentially demonstrating patterns suggestive of self-polymerization. All 15 µM
samples (n=3) were placed in tubulin polymerization buffer containing 1 mM GTP at
37°C using an analytical wavelength of 340 nm to observe polymerization kinetics.
(A) Relative to a bovine tubulin control, TαβS96 similarly exhibits microtubule
dynamics. TαS96 and TβS96 can also self-polymerize in isolation. (B) 15 µM bovine
tubulin data replotted on a separate scale to enhance visualization of its microtubule
dynamics.
A
B
120
Surprisingly, 15 µM TαS96 and 15 µM TβS96 apparently appear capable of
polymerizing at 37°C as ELP-based monomers even in the absence of their respective
cognate partners, a property that has never been reported in the tubulin literature for
either αI-tubulin or βI-tubulin to the best of our knowledge. One previous report in the
literature, however, reveals that γ-tubulin is capable of self-polymerizing. Our initial
supposition was that the S96 tag aided in catalyzing tubulin polymerization, but this
hypothesis stands in direct contravention to previous data demonstrating that neither αI-
tubulin or βI-tubulin exhibited any changes in size or morphology below 40°C. As such,
we theorize that the presence of polymerization-enabling buffer (e.g. GTP, glycerol) in
tandem with the dense number of moieties compacted within each tubulin fusion as
previously determined via SEC-MALS together conspire to permit the polymerization of
monomeric TαS96 and TβS96. Finally, given the fact that data for the 15 µM bovine
tubulin control was obscured by the larger y-axis needed to capture the optical density
profiles for the tubulin fusions, bovine tubulin was segregated into its own
polymerization graph where it becomes readily apparent that – a) bovine tubulin lacks
the nucleation phase seen with the tubulin fusions; and b) even after reaching its Vmax,
the bovine tubulin control exhibits a significantly lower optical density relative to the
tubulin fusions.
121
Having determined the polymerization competence of TαβS96, we next sought to
determine whether these tubulin fusions would respond to paclitaxel, a prototypic
microtubule inhibitor which acts as a stabilizer by arresting microtubule formation and
effectively preventing depolymerization. Interestingly, TαβS96’s polymerization curve
reveals that 10 µM paclitaxel had no demonstrable effect on its polymerization kinetics
in comparison to the bovine tubulin control which exhibited an appreciable shift in its
optical density profile following the addition of paclitaxel (Figure 28).
Figure 28. Paclitaxel does not seemingly enhance the polymerization kinetics
of TαβS96. All samples (n=3) were placed in tubulin polymerization buffer containing
1 mM GTP at 37°C using an analytical wavelength of 340 nm to observe
polymerization kinetics. (A) 5 µM TαβS96 exhibited no difference in its
polymerization kinetics when mixed with 10 µM paclitaxel. (B) As a positive control,
20 µM bovine tubulin demonstrated increased absorbance in the presence of 10 µM
paclitaxel as a microtubule stabilizer.
A
B
122
Although paclitaxel proved incapable of altering TαβS96 polymerization kinetics, the
next set of experiments aimed to deduce whether colchicine and three semisynthetic
colchicinoids ― CBA, CDA, CMA (Figure 2) ― might conversely act as destabilizers
and halt polymerization kinetics in vitro (Figure 29)(Dong et al., 2016). Varying
concentrations (e.g. 0, 5, 10, and 25 µM) of the respective colchicinoid were employed
— ceteris paribus with regard to the experimental conditions of 5 µM TαβS96, 37°C, 30
minutes total, and 400 nm as the selected wavelength — but none of the investigated
ligands demonstrated any concentration-dependent decrease of TαβS96 optical density
Figure 29. Colchicine and three semisynthetic analogues appear incapable of
altering TαβS96 polymerization kinetics. 5 µM samples of TαβS96 samples were
mixed with varying colchicinoids at 0, 5, 10, and 25 µM (n=3) in tubulin
polymerization buffer containing 1 mM GTP at 37°C using an analytical wavelength
of 400 nm to observe polymerization kinetics. (A) CBA; (B) CDA; (C) CMA; and (D)
Colchicine.
A
B
C D
123
as would be expected for a microtubule destabilizer. In the case of the semisynthetic
colchicinoids, in fact, their presence at higher concentrations unexpectedly increased
the optical density profile of TαβS96.
20 µM Bovine tubulin was likewise subjected to destabilization ― marked in optical
density-based assays by delayed polymerization followed by gradual recovery of
polymerization competence (Gigant et al., 2009)― after mixture with colchicinoids at
fixed concentrations of 10 µM (Figure 30). Based on the eventual recovery in optical
density at ~20 min after a period of arrested polymerization kinetics, CBA, CDA, CMA
appear capable of inducing depolymerization. One confounding finding from this study,
Figure 30. Colchicine and three semisynthetic analogues induce
destabilization followed by polymerization recovery in the presence of
bovine tubulin. 20 µM bovine tubulin samples were mixed with 10 µM of varying
colchicinoids in tubulin polymerization buffer containing 1 mM GTP at 37°C using
an analytical wavelength of 400 nm to observe polymerization kinetics. 20 µM
bovine tubulin in the absence of any ligand serves as a positive control.
124
however, was the fact that all colchicinoids examined had optical density profiles which
exceeded the Bovine tubulin positive control lacking any ligand.
Tubulin fusion nanoparticles predominantly appear as oligomers when examined
via negative TEM
Figure 31. Negative TEM reveals that TαS96 exists predominantly as high
aspect ratio nanoparticles. (A) 10 µL of 1 µM TαS96 in ddH2O under non-
polymerizing conditions. Sample was stained with 10 µL of 2% uranyl acetate. A
dominant species of high aspect ratio nanoparticles with average lengths of 130.5 ±
95.4 nm was observed alongside a minor population of spherical structures with an
average diameter of 29.0 ± 19.6 nm. (B) The distribution of particle dimensions for
high aspect ratio TαS96 nanoparticles. (C) The distribution of particle diameters for
spherical TαS96 nanoparticles. Scale bar represents 100 nm.
125
Figure 32. Negative TEM reveals that TβS96 exists predominantly as high aspect
ratio nanoparticles. (A) 10 µL of 1 µM TβS96 in ddH2O under non-polymerizing
conditions. Sample was stained with 10 µL of 2% uranyl acetate. A dominant species
of high aspect ratio nanoparticles with average lengths of 84.8 ± 53.4 nm was
observed alongside a minor population of spherical structures with an average
diameter of 27.0 ± 15.4 nm. (B) The distribution of particle dimensions for high aspect
ratio TβS96 nanoparticles. (C) The distribution of particle diameters for spherical
TβS96 nanoparticles. Scale bar represents 100 nm.
126
Figure 33. Negative TEM demonstrates that Bovine Tubulin
exists as spherical particles. (A) 10 µL of 1 µM Bovine Tubulin
in ddH2O under non-polymerizing conditions. Sample was stained
with 10 µL of 2% uranyl acetate. A population of spherical
particles with an average diameter of 12.5 ± 45.0 nm was
observed. (B) The distribution of particle diameters for Bovine
Tubulin spheres. Scale bar represents 100 nm.
127
Negative transmission electron microscopy
was employed to obtain further information
concerning the structural morphology of
the two tubulin fusions within a non-
polymerizing milieu. Calculated shape
factor data gleaned from SEC-MALS and
DLS provided evidence that the tubulin
fusions likely adopt a prolate ellipsoid
morphology in solution, and this
supposition is supported by the structural
features observed in electron micrographs
for both TαS96 (Figure 31A) and TβS96
(Figure 32A) at 1 µM. SEC-MALS data
also predicted that TαS96 (~1,704
moieties) and TβS96 (~553 moieties)
might exist as densely compacted nanoparticles, based on their high coordination
numbers, which is likewise confirmed by the oligomeric clusters found via negative TEM
imaging. More specifically, a dominant species of high aspect ratio TαS96 nanoparticles
with lengths of 130.5 ± 95.4 nm was observed (Figure 31B) alongside a minor
population of spherical structures exhibiting a diameter of 29.0 ± 19.6 nm (Figure 31C).
While seemingly smaller in size than its counterpart, TβS96 likewise possessed a
dominant species of high aspect ratio nanoparticles with lengths of 84.8 ± 53.4 nm
(Figure 32B) while a minor population of spherical structures exhibited a diameter of
Figure 34. Negative TEM micrograph
of a uranyl acetate-stained blank
grid. A blank grid was stained with 10
µL of 2% uranyl acetate as a negative
control. Scale bar represents 100 nm.
128
27.0 ± 15.4 nm (Figure 32C). For comparison, 1 µM Bovine tubulin was similarly placed
on a negative-stained TEM grid under non-polymerizing conditions to obtain a
population of spherical particles with an average diameter of 12.5 ± 45.0 nm (Figure
33A,B). A blank negative-stained TEM grid was, meanwhile, employed as a negative
control (Figure 34). Quantitative data from these negative TEM studies have been
summarized (Table 12).
129
Table 12. Quantification of Negative TEM Data from Figures 31 to 33.
Structure
a
Average
Length
(nm)
b
Average
Width
(nm)
c
Average
Diameter
(nm)
d
Average
Circularity
(%)
e
Average
Aspect
Ratio
High Aspect
Ratio
TαS96
130.5 ± 95.4 59.5 ± 32.4
f
N/A 47.6 ± 17.8 2.2 ± 0.8
Spherical
TαS96
N/A N/A 29.0 ± 19.6 91.2 ± 7.8 1.3 ± 0.2
High Aspect
Ratio
TβS96
84.8 ± 53.4 38.5 ± 30.1 N/A 37.2 ± 0.2 2.4 ± 0.9
Spherical
TβS96
N/A N/A 27.0 ± 15.4 80.7 ± 8.5 1.4 ± 0.3
Bovine
Tubulin
N/A N/A 12.5 ± 45.0 77.6 ± 24.3 1.7 ± 0.6
a,b
Average length and average length reported as mean ± SD
c
Average diameter obtained from area based on 𝑟 = √
𝐴 𝜋 and D = 2r then reported as
mean ± SD
130
d
Average
circularity determined based on 4𝜋 𝑥
𝐴𝑟𝑒𝑎 [𝑃𝑒𝑟𝑖𝑚𝑒𝑡𝑒𝑟 ]
2
and multiplied by 100 then
reported as mean ± SD
e
Average aspect ratio determined based on
𝐿𝑒𝑛𝑔𝑡 ℎ
𝑊𝑖𝑑𝑡 ℎ
then reported as mean ± SD
f
N/A refers to any measurements which are not applicable
131
Microtubules derived from αβ-tubulin fusions are not apparently visualized via
negative TEM under polymerizing conditions
Figure 35. Negative TEM micrograph reveal that
unpolymerized bovine tubulin is the predominant
population even with paclitaxel present. (A) 1 µM
Bovine Tubulin under polymerization conditions (PME
buffer and 1 mM GTP) in the presence of 10 µM
paclitaxel (n=3). Samples were stained with 2% uranyl
acetate following overnight incubation at 37°C in PME
buffer and GTP to aid formation of paclitaxel-stabilized
microtubules at room temperature for imaging. A
dominant species of spherical structures with average
diameter of 10.9 ± 7.7 nm was identified. (B) The
distribution of particle diameters for spherical bovine
tubulin. Scale bar represents 100 nm.
132
While the prior tubulin polymerization assays offered evidence regarding the apparent
inability of tubulin fusions to respond to ligands, we nevertheless sought to confirm this
discrepancy using negative TEM as an alternative method. As a positive control, 1 µM
Bovine tubulin was incubated overnight under polymerization conditions (PME buffer, 1
mM GTP, 37°C water bath) in the presence of 10 µM paclitaxel to permit the formation
of microtubules for negative TEM visualization (n=3). Prior studies have established that
paclitaxel-stabilized microtubules can remain stable for ~72 hrs at room temperature to
facilitate imaging of these otherwise dynamically unstable structures(Moores, 2008).
Although paclitaxel was added at a 10-fold excess relative to bovine tubulin,
Figure 36. Bovine tubulin forms paclitaxel-stabilized
microtubules under polymerization conditions. 1 µM Bovine
Tubulin under polymerization conditions (PME buffer and 1 mM
GTP) in the presence of 10 µM paclitaxel (n=3). Microtubules
were observed across three different grids (A, B, C). Samples
were stained with 2% uranyl acetate following overnight
incubation at 37°C in PME buffer and GTP to aid formation of
paclitaxel-stabilized microtubules at room temperature for
imaging. Microtubules exhibited an average width of 24.4 ± 0.9
nm. Scale bar represents 100 nm.
133
unpolymerized bovine tubulin remained the predominant population observed with an
average diameter of 10.9 ± 7.7 nm (Figure 35A,B). Nevertheless, paclitaxel-stabilized
microtubules derived from bovine tubulin were visualized across three different grids
(n=3) and exhibited an average width of 24.4 ± 0.9 nm which comports with the ~25 nm
Figure 37. In the presence of paclitaxel, TαβS96 does not apparently form
microtubules. (A) Even under polymerization conditions (PME buffer, 1 mM GTP,
37°C) in the presence of 10 µM paclitaxel, 1 µM TαβS96 nanoparticles do not
assemble into microtubules (n=3). Samples were stained with 2% uranyl acetate
following overnight incubation at 37°C in PME buffer and GTP to aid formation of
paclitaxel-stabilized microtubules at room temperature for imaging.
A dominant species of high aspect ratio TαβS96 nanoparticles with average
lengths of 46.7 ± 57.8 nm was observed alongside a minor population of spherical
structures with an average diameter of 5.2 ± 4.2 nm. (B) The distribution of particle
dimensions for high aspect ratio TαβS96 nanoparticles. (C) The distribution of
particle diameters for spherical TαβS96 nanoparticles. Scale bar represents 100
nm.
134
diameter(Waterman-Storer, 1998) widely reported in tubulin literature (Figure 36).
TαβS96, by contrast, was incapable of forming the rod-like morphology typical of
microtubules following overnight incubation under polymerization conditions (PME
buffer, 1 mM GTP, 37°C water bath) in the presence of 10 µM paclitaxel (Figure 37A).
Similar to TαS96 and TβS96, however, a dominant species of high aspect ratio TαβS96
nanoparticles with average lengths of 46.7 ± 57.8 nm was observed (Figure 37B)
alongside a minor population of spherical structures possessing an average diameter of
5.2 ± 4.2 nm (Figure 37C). Quantitative data from these negative TEM studies have
been summarized (Table 13).
135
Table 13. Quantification of Negative TEM Data from Figures 35 to 37.
Structure
a
Average
Length
(nm)
b
Average
Width
(nm)
c
Average
Diameter
(nm)
d
Average
Circularity
(%)
e
Average
Aspect
Ratio
Spherical
Bovine
Tubulin +
Paclitaxel
f
N/A N/A 10.9 ± 7.7 77.7± 21.3 1.8 ± 0.6
High Aspect
Ratio
TαβS96 +
Paclitaxel
46.7 ± 57.8 23.1 ± 38.6 N/A 44.9 ± 19.0 2.5 ±1.3
Spherical
TαβS96 +
Paclitaxel
N/A N/A 5.2 ± 4.2 94.3 ± 7.6 1.4 ± 0.4
a,b
Average length and average length reported as mean ± SD
c
Average diameter obtained from area based on 𝑟 = √
𝐴 𝜋 and D = 2r then reported as
mean ± SD
d
Average
circularity determined based on 4𝜋 𝑥
𝐴𝑟𝑒𝑎 [𝑃𝑒𝑟𝑖𝑚𝑒𝑡𝑒𝑟 ]
2
and multiplied by 100 then
reported as mean ± SD
136
e
Average aspect ratio determined based on
𝐿𝑒𝑛𝑔𝑡 ℎ
𝑊𝑖𝑑𝑡 ℎ
then reported as mean ± SD
f
N/A refers to any measurements which are not applicable
A kinetics-based proteolytic treatment cleaves TβS96 more effectively than TαS96
or TαβS96
The inability of TαβS96 to respond either to microtubule stabilizers (i.e. paclitaxel) or
destabilizers (i.e. colchicinoids) led us to pivot towards exploring the feasibility of
proteolytically cleaving the S96 fragment via pronase treatment as a means of obtaining
pure αI-tubulin and βI-tubulin monomers. Pronase is a mixture of several nonspecific
endoproteases and exoproteases capable of digesting proteins down to single amino
acids. Prior studies, however, have demonstrated that tubulins can be exposed to
'controlled' proteolysis prior to conducting SDS-PAGE and polymerization to deduce
how the cleavages affect the dimeric protein overall(Sackett and Wolff, 1986; Serrano et
al., 1988). Our rationale is therefore to determine whether pronase can preferentially
cleave S96 as a simple random coil comprised of iterative ELP motifs before
subsequently engaging in cleavage of the more complex tubulin protein structures. To
that end, we sought to determine whether kinetically controlled proteolytic activity could
completely remove the ELP C-terminus and enhance the pool of free tubulin available
for downstream applications.
137
Figure 38. Pronase cleaves TβS96 more effectively than TαS96 or TαβS96.
Proteolytic cleavage of S96 from (A) TαS96; (B) TβS96; and (C) TαβS96 revealed
that a 50 kDa band likely corresponding to βI-tubulin increased concomitantly up to
20 min with a decrease in TβS96 band intensity. TαβS96, by contrast, exhibited the
presence of multiple bands corresponding to various cleavage sites while TαS96
completely dissipated by 20 min. 10 µM tubulin-ELPs samples were mixed 1:1 with
either PBS as a control (Lane 1) or 5 μg/mL pronase at 30°C across 2 min (Lane 2),
5 min (Lane 3), 10 min (Lane 4), 20 min (Lane 5), 30 min (Lane 6), 45 min (Lane 7),
and 60 min (Lane 8) time points. Pronase cleavage was halted at the specified time
point by adding 1 µL PMSF and placing the mixture at 4°C prior to SDS-PAGE
analysis.
C
A B
138
In order to perform proteolytic cleavage, a 1:1 ratio of 10 µg / mL Pronase with 20 µM of
the requisite tubulin fusion (TαS96, TβS96, or TαβS96) was prepared such that the final
concentrations were 10 µM tubulin fusion sample and 5 µg / mL Pronase for 30°C
incubation for the specified amount of time (i.e. PBS blank, 2, 5, 10, 20, 30, 45, or 60
min). Afterwards, pronase activity was halted with the addition of 1 µL PMSF directly to
the reaction mixture followed by being placed on ice prior to SDS-PAGE analysis.
Based on the obtained results, however, it appears that pronase induced such prolific
proteolysis that our strategy involving time-dependent cleavage as a means of removing
S96 provided equivocal results. A smaller band indicative of self-cleavage emerges
within the 100 kDa band corresponding to TαS96 at the 5 min timepoint before TαS96
disappears completely by 20 min (Figure 38A). As the ~100 kDa TβS96 band intensity
gradually decreases, on the other hand, a single 50 kDa band likely corresponding to
free βI-tubulin increases concomitantly up to 20 min (Figure 38B). However, this
pronounced 50 kDa band intensity also dissipates completely by the 30 min timepoint
which is indicative of the transitory nature of this cleavage product. Finally, the
combined presence of TαβS96 was expected to confer some protective benefit from
proteolytic cleavage thereby facilitating more cleavage of the exposed S96 C-terminus;
while this supposition is supported by the fact that the TαβS96 band never completely
dissipates from the SDS-PAGE gel, unlike its monomeric counterparts, multiple
cleavage bands nevertheless appear between the 50 kDa and 75 kDa markers at the 5
min timepoint and persist until the cessation of this experiment (Figure 38C). Overall,
this study establishes that pronase is too promiscuous as an enzymatic cocktail to
provide the tailored cleavage necessary to remove S96 and yield monomeric tubulins.
139
A mammalian-expressed tubulin fusion integrates into the microtubule network
Having investigated TαS96 and
TβS96, we next embarked on a
proof-of-concept study by
engineering a mammalian
vector capable of expressing
TβV72, a hydrophobic ELP
linked to βI-tubulin capable of
forming coacervate-based
microdomains in cellulo (Table
2). Following successful
subcloning into the pcDNA3.1
vector, TβV72 was transfected
into HEK293T cells for 72 hrs at
37°C prior to paraformaldehyde
fixation and immunostaining
with a mouse anti-myc antibody
to facilitate epifluorescence
imaging. Interestingly, TβV72
intercalated into the
cytoskeletal network during interphase as well as mitosis which suggests that 37°C is
below this tubulin fusion’s expected Tt (Figure 39).
Figure 39. A mammalian expressed β-tubulin
ELP fusion intercalates into an existing
microtubule network. TβV72 was transfected into
HEK293T cells, fixed, and detected via anti-myc
antibody using secondary immunofluorescence
prior to epifluorescence imaging. This tubulin
fusion integrated into the microtubule network
where it can be visualized within the mitotic spindle
during (A) mitosis and as (B) cellular scaffolding
during interphase (n=3).
A
B
140
Chapter 4
Discussion and Future Directions
4.1 ELPs as colchicine nanocarriers
Ligands with the capacity to bind tubulins and disrupt the microtubule network represent
an indispensable drug class in the clinical armamentarium of anti-cancer agents. The
recent recombinant expression of human αI/βI tubulins in E. coli by the Tuszynski
laboratory(Mane et al., 2013) therefore provides a useful precedent in synthetic biology
not only for screening potential microtubule inhibitors but also to explore novel drug
delivery approaches leveraging existing FDA-approved drugs as nanomedicines.
Approved in 2009 to treat acute gout flares and familial Mediterranean fever, colchicine
inhibits microtubule assembly by binding β-tubulin at the colchicine-binding site within
the protein’s intermediate region, introducing steric hindrance with α-tubulin during
dimerization, and effectively destabilizing microtubule polymerization. Encouragingly,
two previous studies further established that isolated β-tubulin monomers are sufficient
for colchicine binding. Given the drug’s low therapeutic index due to pronounced and
sometimes fatal off-target toxicities, though, colchicine is not employed as a
chemotherapeutic(Labib et al., 2014). Furthermore, regulatory agencies have yet to
approve any ligand that acts by binding within the colchicine binding site for anti-cancer
applications. The advent of metronomic chemotherapy as a treatment modality,
however, provides an alternative to conventional chemotherapy regimens administered
at maximum tolerated doses in favor of frequent yet low dosing (e.g. 1/10
th
-1/3
rd
of
141
maximum tolerated doses) without drug-free periods(Cazzaniga et al., 2017; Di
Desidero et al., 2016; Kareva et al., 2015); this strategy can be further augmented by
employing nanomedicine-based drug carriers aimed at improving biodistribution,
minimizing off-target effects, and ultimately reducing the total accumulated dose.
Two different ELP fusion proteins derived from human βI-tubulin were genetically
engineered with the goal of exploiting high affinity binding to colchicine as a means of
expanding the MacKay laboratory’s cognate receptor-based nanocarrier strategy
against triple-negative breast cancers. The βI-tubulin amino acid sequence was fused at
its C-terminus to 96 ELP motifs containing Xaa=Serine to form the hydrophilic
monoblock TβS96 as a soluble drug carrier across physiologically relevant
temperatures. TβSI, an amphipathic diblock copolymer expected to form micellar
nanostructures, was similarly constructed as βI-tubulin followed at its C-terminus by 48
motifs of Xaa=Serine and another 48 motifs of Xaa=Isoleucine. Protein expression in
SHuffle cells, an E. coli strain engineered to express disulfide bond isomerase DsbC,
was necessary to obtain the two fusions in a soluble form since βI-tubulin contains eight
cysteine residues capable of forming disulfide bonds critical to proper folding, drug
binding, and microtubule assembly(Chaudhuri et al., 2001); moreover, this effectively
bypasses the labor-intensive protocols typically employed to isolate bacterially
expressed tubulins from inclusion bodies. Inverse thermal cycling of TβSI and TβS96 as
a purification strategy not only yielded high purity material for downstream investigations
but also provided initial evidence ipso facto that these two tubulin fusions were
successfully functionalized with their respective ELP partners. Studies exploring the
142
physicochemical attributes and biophysical properties of the βI-tubulin ELPs revealed
that, despite possessing similar molecular weights based on SDS-PAGE under
denaturing conditions alongside comparable secondary structures when soluble, TβSI
unexpectedly coacervated at a Tt below room temperature relative to TβS96 which
remained stable when tested across a range from 10 to 40°C due to its significantly
higher Tt. This finding stood in stark contrast to our original assumption when designing
the two nanoformulations – namely, that TβSI would serve as the superior carrier
architecture if colchicine could be encapsulated into TβSI micelles to permit an initial
burst release phase followed by the sustained release of bound drug from the micellar
corona. TβS96, on the other hand, was expected to act solely through high affinity
binding of colchicine.
Even after accounting for SI as its ELP fusion partner, TβSI’s more pronounced
thermosensitivity still cannot be entirely explained without recourse to additional data
since SI possesses two characteristic phase transitions ― the critical micelle
temperature and bulk ELP coacervation ― whereas TβSI exhibits its sole phase
transition at ~18°C. Moreover, both TβS96 and TβSI were found to remain stable in
solution with pronounced optical density absorbance profiles relative to ELP controls
below Tt. Examining hydrodynamic radii at 10°C revealed that the tubulin fusions were
~60 nm nanoparticles, and when paired with absolute molar mass, these nanostructures
were both found via their calculated coordination numbers to be richly compacted with
>300 βI-tubulin moieties per nanoparticle. Interestingly, despite existing in a
coacervated state, TβSI also possessed less densely packed fractions consisting
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primarily of dimers. Based on this evidence in toto, we conclude that βI-tubulin ELPs
form preassembled nanostructures regardless of ELP backbone, a finding we have
previously reported with earlier fusion proteins derived from disintegrin(Janib et al.,
2014a), lacritin(Wang et al., 2014b), and anti-CD20 scFv(Aluri et al., 2014). Akin to the
fusion between hydrophilic A192 and anti-CD20 scFv, S96 as a hydrophilic random coil
within the TβS96 configuration is incapable of driving de novo nanoparticle assembly.
LSI, a micellar fusion of lacritin, similarly offers an apposite comparison for TβSI given
their shared SI backbone and proximate phase transitions at ~18°C coupled with LSI’s
preassembly into smaller 30-40 nm nanostructures. Since both tubulin fusions form
preassembled nanostructures, a likely explanation for this phenomenon is that tubulin
moieties amass together into a core buttressed by noncovalent intermolecular forces,
hence the presence of stable ~60 nm nanoparticles as colloids not prone to flocculation
below Tt.
Similar to ELP fusions of lacritin and anti-CD20 scFv, which nevertheless retained
bioactivity despite the presence of nanoparticle cores, we also reported tubulin fusion
bioactivity after observing fluorescence quenching of βI-tubulin tryptophans by
colchicine in a concentration-dependent manner. Due to their intrinsic fluorescence
emission, tryptophan residues act as sensitive reporters of their microenvironment
(Chen and Barkley, 1998). Two tryptophan residues colocalized near the colchicine
binding site (Li et al., 2017; Lu et al., 2012) within β-tubulin’s intermediate region likely
serve as the ligand binding site. In the case of βI-tubulin, which contains 16 tryptophans,
the ability of colchicine to quench intrinsic tubulin fluorescence via forster resonance
144
energy transfer during complexation can effectively be exploited as an experimental
proxy to detect drug binding across emission spectra near 335 nm. Interestingly, TβSI
exhibited concentration-dependent quenching akin to TβS96 despite being assayed
within its coacervated state at 25°C. Quantitative binding data could not be reliably
extrapolated, however, due to - a) the high optical density absorbance values reported
for both tubulin fusions when tested at a fixed concentration of 1 µM due to the likely
nanoparticle core architecture; and b) the high optical density absorbance values
observed when titrating colchicine at excitation λ = 295 nm with an expected emission
λmax = 335 nm to assay tryptophan quenching. Despite attempts to rectify the latter
discrepancy using equations that correct for the inner filter effect, a common issue seen
in fluorescence spectroscopy where concentrated fluorophores absorb and distort
spectral measurements, even low concentrations of our various colchicine titrations
confounded the derivation of a meaningful dissociation constant(Chen et al., 2018).
Although binding between colchicine and tubulin intrinsically quenches the
macromolecule’s tryptophan fluorescence, another biophysical phenomenon occurring
simultaneously involves ligand fluorescence at excitation λ = 350 nm and emission λmax
= 435 nm with its tropolone C-ring acting as the requisite fluorophore(Bhattacharyya et
al., 2010). This dynamic spectral overlap between quenched tubulin tryptophan
emission at 335 nm and emerging colchicine fluorescence at 350 nm can result in the
reabsorption of emitted light known as the secondary inner filter effect; as such, this
dominant yet inevitably confounding factor might account for the inability to derive
reproducible KD values despite the apparent quenching patterns observed in the data.
As a label-free assay relying on the thermodynamic parameters measured during
145
ligand-macromolecule binding, isothermal titration calorimetry might provide an
alternative means of determining KD between colchicine and the tubulin
fusions(Gaisford, 2016).
Given the overall solubility and enduring stability of TβS96 across a broad range of
temperatures relative to TβSI, which conversely coacervates below room temperature,
TβS96 was chosen as the appropriate nanoformulation for investigation as a potential
colchicine-based nanocarrier. A drug retention study, however, invalidated this
supposition as a ~1:1 admixture of TβS96 and colchicine proved incapable of
enhancing drug residence time to any appreciable extent over a free colchicine control.
The fact that our TβS96 nanoformulation was unable to act as a cognate receptor and
retain colchicine despite its reported ability to bind β-tubulin may be due to a confluence
of factors – namely, a) colchicine naturally existing as a water-soluble small molecule
thereby rendering it a poor candidate for drug delivery unless encapsulated which
TβS96 may be incapable of accomplishing due to its tubulin-based core architecture; b)
colchicine’s reported KD = 1.4 µM(Tahir et al., 2000) potentially being too weak to
remain sustainably bound in tandem with a cognate receptor; and c) our C-terminal
tagging of βI-tubulin with S96 possibly abolishing tubulin’s ability to bind and/or retain
drug since the variable sequences of disordered C-terminal tails serve to distinguish
tubulin isotypes and regulate their various intracellular functions(Laurin et al., 2017;
Parker et al., 2018). Although the colchicine binding site is located inside tubulin’s
globular core within the intermediate region, one can speculate that modification of the
critical C-terminal tail might induce non-contiguous yet global effects throughout the
146
protein thus influencing its sensitivity to ligands. While circular dichroism assayed within
the far-ultraviolet region can yield secondary structural data, this technique cannot
provide the tertiary information critical to predicting drug binding unless assayed at the
near-ultraviolet region. In the absence of α-tubulin, however, ruling out the binding
competence of β-tubulin ELPs remains conjecture, hence additional studies were later
undertaken with dimeric αβ-tubulin ELPs.
As a final note concerning TβSI, while our goal was to develop a thermosensitive
nanocarrier capable of being loaded in a soluble state with colchicine prior to phase
transition for generation of an extended drug depot, its Tt below room temperature
curtails further exploration unless investigators are willing to conduct preparatory work
on ice and all experimental studies under temperature-controlled settings. The most
appropriate means of salvaging the concept of cognate tubulin receptors as a
thermoresponsive drug depot involves reengineering βI-tubulin such that any
accompanying ELP partner exhibits a higher Tt. This can be accomplished by
appending ELPs possessing either different and/or hybridized hydrophobic guest
residues (e.g. Valine, 2VA) or shorter lengths corresponding to lower molecular weights
(e.g. V72, V60). Although these biophysical studies have detailed the soluble
expression and physicochemical characterization of βI-tubulin fusions with different ELP
architectures and corresponding thermoresponsive profiles, our work suggests that
recombinant βI-tubulin cannot be exploited as a cognate receptor to facilitate prolonged
colchicine retention through the techniques investigated.
147
4.2 ELPs as purification tools
Having established ELP-mediated inverse thermal cycling as a facile method for the
soluble expression of human βI-tubulin from E. coli, we sought to broaden the scope of
this novel purification strategy by engineering an α-tubulin ELP fusion as a soluble
complement to TβS96 capable of recapitulating microtubule polymerization. The αI-
tubulin amino acid sequence was therefore fused at its C-terminus to 96 ELP motifs
containing Xaa=Serine to generate the hydrophilic monoblock TαS96. SHuffle cells
likewise expressed TαS96 in a readily soluble form despite the fact that αI-tubulin
contains twelve cysteine residues, some of which can produce disulfide
bonds(Chaudhuri et al., 2001). Owing to their shared S96 backbone, both tubulin
fusions remain soluble across physiologically relevant temperatures while also
possessing ― mutatis mutandis ― comparable molecular weights and secondary
structures. Interestingly, TαS96 exhibited an approximately 20°C reduction in Tt relative
to TβS96 alongside a more robust correlation between increased concentrations and
decreasing temperatures. One parsimonious explanation arises following a differential
analysis of the hydrophobic amino acid residues comprising αI-tubulin and βI-tubulin
respectively. Although both proteins share a high degree of homology based on their
near equivalent number of most hydrophobic residues, βI-tubulin nevertheless
possesses 17 methionine residues in comparison to 10 methionine residues for αI-
tubulin. Conversely, αI-tubulin contains 27 isoleucine residues relative to the 19
isoleucine residues found in βI-tubulin. Another interpretation relies on data revealing
148
that TαS96 preassembles into core nanostructures akin to βI-tubulin ELPs. TαS96 was
found to have larger hydrodynamic radii than TβS96 and congregated into ~1,700 αI-
tubulin moieties per nanoparticle; TβS96, by contrast, was reported to coalesce into
>500 βI-tubulin moieties per nanoparticle. As such, these data collectively yield the
most apposite evidence for TαS96’s diminished Tt relative to its counterpart.
When mixed together at a 1:1 ratio, TαβS96 exhibited concentration-dependent tubulin
tryptophan fluorescence quenching but nevertheless proved less responsive to
increasing colchicine concentrations relative to dimeric bovine tubulin and TβS96 alone.
The significantly larger fluorescence intensities observed for the TαβS96 spectra,
however, likely mask the more pronounced optical density absorbance values resulting
from the tandem presence of TαS96, TβS96, and colchicine in solution; as
aforementioned, high optical density solutions render derivation of dissociation
constants from fluorescence spectroscopy nigh impossible due to the secondary inner
filter effect. Absorbance-based polymerization studies conducted at λ = 340 nm in the
presence of GTP subsequently revealed that TαβS96 exhibited the three characteristic
phases of polymerization kinetics ― nucleation, growth, and steady state equilibrium ―
typical of dimerized tubulin. The high optical density profiles observed for TαβS96
relative to a dimeric bovine tubulin control are indicative of intense light scattering likely
resulting from assembly of these large tubulin core nanostructures together.
Interestingly, TαS96 and TβS96 exhibited self-polymerization separately in
polymerization buffer which represents a novel finding in the tubulin literature to the best
of our knowledge. To confirm this supposition, however, an alternative method should
149
be further pursued to yield independent confirmation. While fluorescence-based tubulin
polymerization assays using DAPI(Arbildua et al., 2006) might serve as a secondary
means of confirming polymerization, the unique properties of tubulin-ELPs might
confound any meaningful derivation akin to the secondary inner filter effect observed
during fluorescence spectroscopy measurements. As such, a more reliable method
might be the scintillation proximity assay employing biotinylated tubulin as a reagent in
tandem with tritiated colchicine as a radio-ligand to generate a signal that is directly
proportional to the number of colchicine binding sites being occupied to demonstrate
concentration-dependent polymerization destabilization(Tahir et al., 2000).
Visualization via negative transmission electron microscopy, meanwhile, revealed that
TαS96 and TβS96 both clustered into oligomers in the absence of polymerization buffer.
A recent study (Chumova et al., 2018)demonstrating that γ-tubulin, a cognate protein
responsible for microtubule nucleation, has the capacity to self-polymerize in plant and
human sourced material while also forming oligomers in porcine samples offers a
compelling context for parsing these findings. Given that three tubulin-ELPs have been
empirically demonstrated to form de novo nanostructures suffused with tubulin moieties
at their core, we theorize that tubulin’s globular architecture ― comprised of highly
conserved N-terminal and intermediate regions ― form nuclei from which ELP-
functionalized C-terminal tails interact to permit noncovalent intermolecular forces
thereby promoting oligomerization reminiscent of γ-tubulin self-polymerization.
Returning to the earlier supposition that drug binding to tubulin-ELPs could not be
150
discounted without testing a dimeric αβ-tubulin ELP, despite the fact that TβS96 proved
incapable of enhancing colchicine retention, we employed tubulin polymerization assays
to investigate TαβS96’s ability to bind two classical microtubule inhibitors (paclitaxel and
colchicine) alongside three semisynthetic analogues of colchicine abbreviated as CBA,
CDA, and CMA (Figure 2)(Dong et al., 2016). Confoundingly, however, TαβS96
polymerization kinetics remained unresponsive to every microtubule inhibitor employed.
Negative transmission electron microscopy also revealed that TαβS96 does not form
rod-like microtubules even when mixed with paclitaxel in polymerization-competent
buffer; the dimeric tubulin-ELP admixture instead exists as a dense agglomeration of
oligomers existing largely as high aspect ratio nanoparticles with a smaller population of
spherical nanoparticles. This is in in line with electron micrographs reported for TαS96
and TβS96 which exhibit the same populations of high aspect ratio nanoparticles
interspersed with smaller spherical nanoparticles. Cryogenic electron microscopy
(CryoEM) might yield additional morphological information since this technique
facilitates the visualization of nanostructures in a near physiological state following rapid
freezing(Moores, 2008). Sample preparation for cryo-EM involves ultra-rapid freezing.
Interestingly, the presence of tubulins possessing reduced sensitivity to microtubule
inhibitors has dire clinical consequences since aggressive cancers typically upregulate
the neuronally expressed βIII-tubulin isotype as a means of drug resistance(Parker et
al., 2017) by effectively increasing the chemotherapy concentrations needed to
suppress microtubule dynamics. In our studies, however, the ubiquitously expressed βI-
tubulin isotype was employed in tandem with αI-tubulin thus precluding the tubulin
isotypes as the cause of abolished drug binding.
151
A more plausible theory was described earlier and can now be more thoroughly
supported ― namely, that C-terminal functionalization of αI-tubulin and βI-tubulin,
respectively, with S96 abrogated tubulin’s ability to bind microtubule inhibitors or
assume the prototypic microtubule architecture. This results from the fact that tubulin C-
terminal tails(Laurin et al., 2017) modulate the varying functions of tubulin isotypes via
post-translational modifications including spatiotemporal coordination of dynamic
instability(Parker et al., 2018) as well as sensitivity to microtubule inhibitors(Dorleans et
al., 2009); alteration of this critical C-terminal tail with an ELP moiety might therefore
account for this observed inability to engage in ligand binding. An alternative hypothesis
is that oligomerization of tubulin-ELPs into densely packed nanostructures, according to
SEC-MALS findings, now render binding of microtubule inhibitors at their requisite
docking location (e.g. colchicine-binding site, taxane-binding site) an impossibility.
Suspecting that the fusion of ELPs at tubulin C-terminal tails might indeed account for
these disparities, we sought to selectively cleave off S96 to obtain purified αI-tubulin, βI-
tubulin, and αIβI-tubulin in a time-dependent manner using a powerful protease cocktail
known as pronase. This proteolytically active admixture, however, proved too non-
specific for the purpose of selectively isolating free tubulins.
Considered collectively, while αβ-tubulin fusions exhibit absorbance-based
polymerization kinetics mimicking native tubulins, ELP functionalization nevertheless
introduces morphological aberrations hindering their ability to be fully realized as
recombinant tubulin surrogates for applications in basic research and as potential
152
microtubule inhibitor screening tools. A parsimonious means of rapidly addressing these
drawbacks and obtaining purified free tubulins involves reengineering tubulin-ELPs such
that a defined cleavage site (e.g. thrombin) is incorporated for proteolytic removal of the
ELP tag following a sufficient number of inverse thermal cycling rounds(Waugh, 2011).
An alternative strategy, meanwhile, involves redesigning the tubulin fusions with the
ELP moiety encoded adjacent to tubulin’s globular N-terminal region. One caveat to
consider, however, is the fact that ELP constructs localized at the N-terminus have been
reported to generate lower yields of the expected fusion protein relative to their C-
terminal ELP counterparts(Christensen et al., 2009).
4.3 ELPs as intracellular switches
As native protein polymers exquisitely regulated in a spatiotemporal manner,
microtubules are key intracellular components of the complex cytoskeletal network and
play a myriad of roles that range from supporting cellular integrity and trafficking to
participating in cell motility and ensuring chromosomal segregation throughout mitosis.
Designing tools derived from synthetic biology capable of modulating this dynamic
system in a rapid and reversible manner in situ relative to traditional pharmacological
administration therefore represents an exciting opportunity not only to further our
understanding of tubulin within its intracellular milieu but also to envision new
applications for microtubules as multifunctional biomolecular machines. While prior
studies investigating microtubule dynamics have largely focused on the passive use of
GFP-tubulins, one group pioneered the LARIAT (light-activated reversible inhibition by
153
assembled trap) strategy to actively control microtubules in HeLa cells(Lee et al., 2014).
This method relies on the use of GFP-tubulin to sequester the mitotic spindle into
optically assembled complexes formed by multimeric proteins and a blue light–mediated
heterodimerization module. Another recent study (Borowiak et al., 2015)employed a
catalogue of photoswitchable microtubule inhibitors called photostatins to induce mitotic
arrest and apoptosis in a light-dependent manner as a means of probing cell division
and potentially serving as high-precision chemotherapeutics. Given its capacity to form
heat-inducible yet rapidly reversible microdomains, ELPs can similarly be positioned as
intracellular tubulin cognates that actively integrate within the microtubule network to
further probe processes such as dynamic instability and mitotic arrest.
The βI-tubulin amino acid sequence was fused at its C-terminus with a 1.2 kDa myc tag
followed by 72 ELP motifs containing Xaa=Valine to generate the hydrophobic
monoblock TβV72 for mammalian expression. Following transient transfection in
HEK293T cells for 72 hours at 37°C, TβV72 was fixed for subsequent immunostaining.
When observed via epifluorescence microscopy, this tubulin fusion intercalated into the
microtubule network during interphase as well as mitosis. Since interphase microtubules
are primarily involved in maintenance of cell shape while also facilitating the trafficking
of proteins and organelles, this might account for the diffuse fluorescence pattern
observed for TβV72 throughout the cell under investigation. In contrast, the microtubule
network reorganizes during mitosis to generate the mitotic spindle with individual
microtubule-based filaments aiding chromosomal segregation, hence the localization of
TβV72 solely within the mitotic spindle.
154
These findings stand in stark contrast to our initial assumption that an excess of free βI-
tubulin might interfere with the pool of cytosolic tubulins by skewing the equilibrium
between heterodimeric and soluble tubulins towards more rapid microtubule
disassembly and subsequent cell death(Matsuzaki et al., 1988). Intriguingly, the fact
that TβV72 was able to integrate into the microtubule network despite possessing an
ELP appended to the critical C-terminal region suggests that ELP orientation may not
prove as much of a drawback in an intracellular milieu as it was in an in vitro setting for
TαS96, TβS96, and TβSI. As a eukaryotic protein, the bacterial expression of tubulin in
the absence of its native cofactors and chaperones might also account for the
experimental difficulties observed. Another interesting finding was that TβV72 did not
undergo phase transition and form observable microdomains at 37°C which is indicative
of the fact that its Tt occurs at a higher temperature. One possible theory is that a
protein’s intracellular Tt might diverge significantly from its expected Tt in vitro, and in
the case of TβV72, its potential interactions with multiple protein partners within a
cellular milieu might induce disparate behaviors. As such, this provides a compelling
rationale not only for employing tools capable of predicting Tt a priori but also for the
design of a mammalian expressed TβSI exhibiting a lower Tt for applications pertaining
to intracellular switching. Since further research must be undertaken to elucidate the
intracellular behavior of TβV72, however, future potential studies with this construct are
detailed below.
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4.3.1 Future Directions
As a means of empirically deducing this tubulin fusion’s transition temperature, cytosolic
GFP-V60 will be co-transfected with TβV72 to provide a fluorescent intracellular tracker
during TβV72 microdomain assembly as established by previous work employing this
strategy with an EGFR-ELP fusion(Li et al., 2018). Based on results using doubly
transfected GFP-V96 and EGFR-V96, we anticipate that GFP-V60 can likewise
colocalize with TβV72. The precision afforded by live cell imaging will allow us to
characterize the kinetics of tubulin microdomain assembly across a temperature ramp in
order to: 1) measure the percentage of cells that transition; 2) identify the morphological
structures of the microdomains formed within the microtubule network; and 3) determine
whether heat-inducible mitotic arrest occurs. Co-transfections of GFP-V96 and Tβ-myc
will be run in parallel as a negative control. Below Tt, TβV72 should freely participate in
dynamic instability since tubulins have been shown to polymerize into microtubules
irrespective of isotype37. Following a temperature ramp past its Tt, however, TβV72 will
likely assemble into microdomains and potentially act as heat-inducible ‘tubulin traps’
capable of disrupting the microtubule network to promote mitotic arrest and apoptosis.
Based on prior epifluorescence microscopy data using fixed cells, it is expected that
TβV72 will have a Tt >37°C.
If TβV72 microdomain formation proves successful in encouraging mitotic arrest, we
expect that various apoptotic hallmarks will be investigated to validate our drug-free
tubulin trap strategy. Cell death induced by microdomain disruption of the microtubule
156
network can be assayed using immunofluorescence followed by fixed cell imaging
against apoptotic targets(Cummings and Schnellmann, 2004). Live cell imaging will,
meanwhile, allow us to study the processes underlying TβV72-induced apoptosis in real
time. Finally, synergies in apoptotic efficacy between TβV72 microdomains and
microtubule inhibitors will be examined. We therefore seek to challenge TβV72 / GFP-
V60 co-transfected TNBC cell lines (e.g. MDA-MB-468, MDA-MB-231) with microtubule
inhibitors and observe via live cell imaging and in situ fluorescein-based TUNEL
whether apoptosis occurs at greater rates once Tt is reached. This study will further
allow us to determine how tubulin isotype switching permits survival in cancer cells. It is
expected that an excess of exogenously introduced TβV72 will prompt native βI-tubulin
downregulation in favor of the βIII-tubulin isotype regularly encountered in drug-resistant
tumor cells. While this survival factor may mitigate the toxicity of microtubule inhibitors,
we still anticipate that TβV72 microdomains will effectively permit apoptosis. Overall, the
available preliminary data provide encouraging evidence that TβV72 might be
applicable as a tubulin-based tool capable of thermally modulating microtubule
dynamics in a spatiotemporal manner; in addition, the unique interactions between ELP-
tagged tubulin and other essential cytoskeletal elements opens the exciting possibility of
eventually recapitulating the mitotic spindle apparatus as an artificial organelle subject
to tunable regulation.
157
4.4 Conclusions
The purpose of this dissertation has been to describe state-of-the-art developments
involving tubulins genetically fused with elastin-like polypeptides to generate
multifunctional tools. Three versatile strategies have therefore been disclosed within
these pages with the intent of leveraging the unique properties of these tubulin fusion
proteins to address various challenges in vitro as well as within an intracellular milieu.
Biophysical studies established that human αβ tubulins can be expressed recombinantly
and purified in a soluble state appended to different ELP architectures, but our work
suggests that recombinant βI-tubulin cannot be exploited as a cognate receptor for drug
delivery to facilitate prolonged colchicine retention through the techniques investigated.
While αβ-tubulin fusions exhibit polymerization kinetics resembling native bovine
tubulin, ELP functionalization apparently introduces morphological aberrations hindering
their ability to be fully realized as potential microtubule inhibitor screening tools. A
tubulin-ELP was additionally engineered as an intracellular tubulin cognate to further
probe processes such as dynamic instability and mitotic arrest. The experimental
approaches investigated throughout this dissertation alongside the corresponding
results have been summarized (Table 14). In closing, despite the fact that these novel
tubulin-based tools necessitate further investigation, we maintain that ELPs might
eventually serve as powerful tools in the tubulin field with the capacity to potentiate new
research avenues while bridging the burgeoning fields of synthetic biology and
nanomedicine.
158
Table 14. Summary of Experimental Approaches and Results.
Experimental
Approach
Proteins
Investigated
Results
Applicable
Datasets
Western Blotting TβSI TβSI expressed in BLR
cells was found to be
sequestered into
insoluble inclusion
bodies within bacterial
pellets
Figure 11
SDS-PAGE TβSI, SI,
TαS96, TβS96,
S96, Human βI-
Tubulin-GST,
Bovine Tubulin
TαS96, TβS96, and
TβSI expressed in
SHuffle cells exhibited
soluble expression with
protein yields of ≥15
mg/L at ≥80% purity
Figure 12, Figure
13, Table 3, Table 4
UV-Vis
Spectroscopy
Temperature
Ramp
TαS96, TβS96,
TβSI, S96, SI
TβSI possesses a Tt
>25°C unlike SI. TαS96
and TβS96 both remain
soluble from 10°C to
40°C akin to S96
Figure 14, Figure
15, Table 5, Table 6
159
Circular
Dichroism
Spectroscopy
TαS96, TβS96,
S96, TβSI, SI,
Bovine Tubulin
TαS96 and TβS96
exhibit far-UV spectra
akin to Bovine Tubulin
which possesses an α-
helical spectrum; S96
and SI possess random
coil spectra
Figure 16
Circular
Dichroism
Deconvolution
TαS96, TβS96,
S96, TβSI, SI,
Bovine Tubulin
The BeStSel model
produces an accurate fit
and permits
quantification of
secondary structural
motifs
Figure 17, Table 7
Dynamic Light
Scattering
TαS96, TβS96,
S96, TβSI,
Bovine Tubulin
TαS96 is ~80 nm in
hydrodynamic radii
relative to ~60 nm for
Bovine Tubulin, TβS96,
and TβSI; S96 exhibits
~5 nm nanoparticles
Figure 18, Figure 19
MALDI-ToF TβS96, S96,
TβSI, SI
The <40 kDa molecular
weights of S96 and SI
are confirmed whereas
Figure 20, Figure 21
160
TβS96 and TβSI yielded
molecular weight
determinations
SEC-MALS Bovine Serum
Albumin,
Bovine Tubulin,
TαS96, TβS96,
S96, TβSI, SI
Bovine Serum Albumin,
Bovine Tubulin, and S96
each eluted as one
monomeric fraction near
their anticipated
molecular weights; SI
eluted as two oligomeric
fractions whereas TβSI
eluted as an oligomeric
fraction followed by two
dimeric fractions; TαS96
and TβS96 each eluted
as one oligomeric
fraction
Figure 22, Figure
23, Figure 24, Table
8, Table 9, Table 10
Fluorescence
Spectroscopy
Bovine Tubulin,
TαβS96,
TβS96, TβSI
Due to the secondary
inner filter effect
promoted by colchicine
fluorescence excitation,
colchicine was found to
Figure 25
161
absorb fluorescence
emitted during tubulin
quenching which
prevented reproducible
derivation of quantitative
data
Colchicine
Retention
TβS96 TβS96 did not prolong
colchicine retention time
via extended dialysis
followed by RP-HPLC
analysis relative to a free
colchicine control
Figure 26
Tubulin
Polymerization
Bovine Tubulin,
TαβS96,
TαS96, TβS96
Akin to Bovine Tubulin,
TαβS96 as well as
TαS96 and TβS96 each
appear capable of
engaging in
polymerization kinetics.
Relative to Bovine
Tubulin, however,
TαβS96 remains
unresponsive to ligand-
Figure 27, Figure
28, Figure 29,
Figure 30
162
based microtubule
inhibitors such as
paclitaxel and
colchicinoids
Negative
Transmission
Electron
Microscopy
Bovine Tubulin,
TαβS96,
TαS96, TβS96
Bovine Tubulin exists as
a population of spherical
particles capable of
assembling into ~25 nm
microtubules in the
presence of paclitaxel.
TαS96 and TβS96
predominantly exist as a
population of high
aspect ratio
nanoparticles that, when
mixed to form TαβS96,
remain incapable of
forming microtubules in
the presence of
paclitaxel
Figure 31, Figure
32, Figure 33,
Figure 34, Figure
35, Figure 36,
Figure 37, Table 11,
Table 12, Table 13
Pronase
Treatment
TαβS96,
TαS96, TβS96
Based on SDS-PAGE,
pronase cleaves TβS96
Figure 38
163
with greater specificity
relative to TαS96 and
TαβS96
Epifluorescence
Microscopy
TβV72 Intracellularly expressed
TβV72 integrates into
the existing microtubule
network where it is
visualized within the
mitotic spindle during
mitosis and as cellular
scaffolding during
interphase
Figure 39
164
Acknowledgements
This dissertation was made possible by the University of Southern California and the
National Institutes of Health R01GM114839 to JAM, the USC School of Pharmacy
Translational Research Laboratory, and the USC Nanobiophysics Core Facility.
List of Abbreviations
AAV – Adeno-associated virus
AntP – Penetratin
BPE – Bovine pituitary extract
CFTR – Cystic Fibrosis Transmembrane Conductance Regulator
Chol – Cholesterol
CMT – Critical micelle temperature
CN – Contortrostatin
CPP – Cell-penetrating peptide
CVD – Cardiovascular disease
DIC – Differential interference contrast
DPPC – 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
DSPE-PEG – 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-
[methoxy(polyethyleneglycol)-2000]
ECM – Extracellular matrix
EGF – Epidermal growth factor
165
ELP – Elastin-like polypeptide
eNOS – Endothelial nitric oxide synthase
EPR – Enhanced permeability and retention effect
FKBP – FK506 binding protein 12
FSI – FKBP-S48I48
ΔG – Change in Gibbs free energy
GLP-1 – Glucagon-like peptide 1
ΔH – Change in enthalpy
H&E – Hematoxylin and eosin
IL-1Ra – Interleukin 1 receptor antagonist
IL-10 – Interleukin 10
ITC – Inverse transition cycling
iTEP – Immune-tolerant elastin-like polypeptides
KGF – Keratinocyte growth factor
KGFR – Keratinocyte growth factor receptor
LSI – Lacritin-S48I48
mAb – Monoclonal antibody
Mn – Number-average molecular weight
mTOR – Mammalian target of rapamycin
166
Mw – Mass-average molecular weight
NOD – Non-obese diabetic
OERCA – Overlap extension rolling circle amplification
OVA – Ovalbumin
PCL – Polycaprolactone
PCR – Polymerase chain reaction
PDI – Polydispersity index
PEG – Polyethylene glycol
PRe-RDL – Recursive directional ligation by plasmid reconstruction
qPCR – Quantitative polymerase chain reaction
ΔS – Change in entropy
scFv – Single chain variable fragment
siRNA – Small interfering RNA
SI – S48I48
SjS – Sjögren’s syndrome
SLP – Silk-like polypeptide
THPP – β-[Tris(hydroxymethyl) phosphine] proprionic acid
TNFα – Tumor necrosis factor alpha
TRAIL – Tumor necrosis factor-related apoptosis-inducing ligand
167
T – Temperature
Tt – Transition temperature
VCN – Vicrostatin
VEGF – Vascular endothelial growth factor
VIP – Vasoactive intestinal peptide
XTEN – Extended recombinant polypeptide
168
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Abstract (if available)
Abstract
This dissertation investigates the prospect of harnessing tubulins genetically fused with elastin‐like polypeptides as multifunctional tools. Tubulins are dynamic proteins which assemble into polymers called microtubules to participate in critical roles throughout the cell such as cytoskeletal stability, intracellular transport, and mitosis. Given their physiological importance, these native protein polymers have been clinically exploited as targets for small molecule drugs capable of interfering with the microtubule network. Elastin‐like polypeptides (ELPs), meanwhile, constitute a genetically engineered class of ‘protein polymers’ derived from human tropoelastin. They exhibit a reversible phase separation whereby samples remain soluble below a transition temperature (Tₜ) but form amorphous coacervates above Tₜ. ELPs phase behavior has many possible applications in purification and nanoassembly which can be further expanded by fusing this thermoresponsive moiety to native proteins. Recombinant synthesis additionally affords precise control over an ELP fusion protein’s architecture and molecular weight, resulting in functionalized protein polymers with uniform physicochemical properties suited to the design of tools relevant to nanomedicine and synthetic biology. The studies featured within this dissertation therefore explore tubulin‐ELP fusion proteins as multifunctional tools with three broad applications—namely, as colchicine nanocarriers, tubulin‐based purification tools, and intracellular switches. Although unsuccessful as colchicine nanocarriers, these ELP‐based tools have potentiated a non‐chromatographic method for the purification of recombinantly expressed tubulins and offer the possibility of thermally regulating microtubule dynamics within an intracellular milieu.
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Despanie, Jordan
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Core Title
Tubulin-based fusion proteins as multifunctional tools
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School of Pharmacy
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Doctor of Philosophy
Degree Program
Pharmaceutical Sciences
Publication Date
04/19/2019
Defense Date
12/20/2018
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biophysics,Colchicine,drug delivery,elastin‐like polypeptides,microtubules,nanomedicine,OAI-PMH Harvest,protein polymers,synthetic biology,tubulin
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Tags
biophysics
drug delivery
elastin‐like polypeptides
microtubules
nanomedicine
protein polymers
synthetic biology
tubulin