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Synthetic biopolymers modulate cell signaling

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Synthetic biopolymers modulate cell signaling

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Content SYNTHETIC BIOPOLYMERS MODULATE CELL SIGNALING
by
Anh Tan Truong
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)
December 2021
Copyright 2021 Anh Tan Truong
ii

DEDICATION

This dissertation is dedicated to my wife Anna and the Kiyomi family.
The Kiyomi family welcomed me as their own and has made this long journey meaningful.

iii

ACKNOWLEDGEMENTS
I want to acknowledge and express my sincere gratitude to both my co-advisor’s Dr. Curtis
T. Okamoto and Dr. J. Andrew MacKay. During my first year as a PharmD graduate student, Dr.
Okamoto introduced me to the PhD program and the ongoing research at USC School of Pharmacy.
He evaluated my research interests and based on his assessment, thought I was a great fit and
introduced me to Dr. MacKay. For the next 7 years where I called my home, Dr. MacKay provided
guidance during this period to become a great scientific communicator. Both Dr. Okamoto and Dr.
MacKay has tremendously accelerated my intellectual thinking, for which I am very grateful. I
have been able to take advantage of these two experts to understand more about the world of
science and used it to mold my career path. Additionally, without these two co-mentors, I may not
have had the opportunity to pursue a PhD at USC. They both advocated for and helped construct
the PharmD/PhD program for which I am part of today.
I also want to thank Dr. Ian Haworth for his full support while in the PharmD and PhD
program. Dr. Haworth secured opportunities for me that would otherwise be inaccessible. From
assisting in his pharmaceutics course, to navigating through obtaining a teaching certificate at
USC; he even helped me acquire my first ‘course coordinator’ position for the international global
initiatives- something I truly wanted to be a part of. Words truly cannot describe my appreciation
for his assistance through my academic career. I also want to show appreciation for Dr. Rebecca
Romero, who encouraged and reinforced the idea of research during my PharmD. Along with Dr.
Romero, I want to show appreciation for Dr. Paul Beringer. When I was considering between a
PhD and a clinical fellowship, his words resonated with me and based on my future goals, a PhD
would be more valuable; this solidified my choice to pursue additional training.
iv

I thank Dr. Sarah Hamm-Alvarez and her laboratory including Francie Yarber, Dr. Mihir
Shah, Dr. Zhen Meng, Dr. Maria Edman-Woolcott and Dr. Pang-Yu (Aaron) Hsueh for showing
me the ropes when I first began research at the School of Pharmacy. Dr. Hamm-Alvarez, who is
part of my committee, provided me a third home where I could openly ask general research and
experimental questions. This nest literally helped me to become accustomed to the academic life
at USC. I would also like to thank my other committee members, Dr. Bangyan Stiles and Dr.
Houda Alachkar, who both had provided constructive comments during my PhD qualification
examination. I have had the pleasure to work along the side both their present and previous
graduate students and understand both of their research through their mentees.
Moreover, I also want to extend my thanks to our collaborators who looked out for my
well-being during my year conducting research abroad in Japan. Dr. Motoyoshi Nomizu who was
my mentor, was instrumental in guiding my research to quickly produce results for publication.
He also assisted me in joining international and local symposiums to meet other scientists. I thank
Dr. Keisuke Hamada for being the key person to help drive my studies in regard to chemistry
synthesis. He also was extremely helpful in general laboratory questions, ideas and with all the
complex Japanese translations. I also thank Dr. Kikkawa Yamato and Dr. Yuji Yamada for their
expert opinion for some of my experiments. Their expertise in the area supported my research
extensively. Additionally, I want to thank Dr. Nobuhito Hamano, Dr. Kenji Onda, Dr. Sachiko
Tanaka, Dr. Shintaro Besshoh and Dr. Katsunori Miyake for their support while overseas.
I thank Dr. Zhe Li who took time out her busy schedule to mentor me and always patiently
answer my experimental questions. Dr. Li is one of the greatest mentors anyone could ask for. For
my other colleagues who have made this PhD experience joyful, I thank, Dr. Mincheol Park, Dr.
David R. Tyrpak, Dr. Jingwen (Julianna) Chen, Dr. Aida Kouhi, Dr. Larry Rodriguez, Dr. Li Zhou,
v

Dr. Hsuan-Yao (Sean) Wang, Dr. Runzhong (Christina) Fu, Dr. Hao Guo, Dr. Santosh Peddi, Yang
(Lisa) Su and soon to be Drs. Hugo Avila and Taojian (Michael) Tu. Ryan van Damme, Wilson
Lee, Minchang Choi, Chris Rabot and Sam Garza are the building members who could always
strike up a conversation within the school building hallways. I could always count on these school
of pharmacy members, whether it be scientific discussions or just plain non-sense. Additionally, I
give thanks to those who have moved or soon will- to advance their careers: Yue Wang, Geetha
Boddu, Zhiyuan Yao, Zeyu (Angel) Zhang, Chenchang (Grace) Liu and Yutong Wang. Without
the support pillar these friends provided, the journey would have been grim.
Finally, I want to thank my friends and other mentors who have supported me in my long
career as a professional graduate student. Dr. J. Brandon White who was my previous mentor-
without his push into biological sciences, I absolutely would not have been here today. My best
friend Edwin Cho who, despite making jokes about my academic career, always recognized my
talent in this field. My high school friends, Viet Nguyen, Tomi and Tera Ngo, Joe Le, Jason and
Jill Cho and Duan Nguyen. These are the friends who would drop their plans to come have dinner
with me when I came back to the bay area on short notice. They have seen my sweat and tears and
have been my second family, urging me to push forward- and for this, I am grateful that while this
journey has come to an end, that another will open.
vi

TABLE OF CONTENTS
DEDICATION ……………………………………………………………………………….… ii
ACKNOWLEDGEMENTS …………………………………………………………………... iii
LIST OF TABLES ……………………………………………………………………………... x
LIST OF FIGURES ………………………………………………………………………….... xi
ABBREVIATIONS …………………………………………………………….…………...... xiv
ABSTRACT …………………………………………………………………………….…...... xvi
CHAPTER 1: INTRODUCTION ……………...….………………………….………….……. 1
1.1.Cell signaling ………………………………………………………………….......... 1
1.2.Thermo-responsive biopolymers …………………………………………………… 2
CHAPTER 2: EXTRACELLULAR MATRIX MIMETICS TO PROMOTE CELL
ADHESION AND ATTACHMENT……………………………………………………………5
2.1. Abstract……………………………………………………………………………….5
2.2. Introduction…………………………………………………………………………...6
2.3. Material and Methods………………………………………………………………...9
2.3.1. Reagents…………………………………………………………………….9
2.3.2. Construction, expression and purification of ELPs………………………...9
2.3.3. Determining the lower critical solution temperature……………………...11
2.3.4. Peptide synthesis and conjugation………………………………………...11
2.3.5. Cell culture………………………………………………………………...12
2.3.6. Dose-dependent assay……………………………………………………..12
2.3.7. Cell attachment and spreading assay……………………………………...13
2.3.8. Immunofluorescence staining and fluorescence microscopy……………..14
2.3.9. Statistics…………………………………………………………………...15
2.4. Results……………………………………………………………………………….15
2.4.1. Generation of thermo-responsive bioactive laminin peptide-ELPs……….15
2.4.2. Protein purification and characterization……………………………….....17
vii

2.4.3. Chemical synthesis and conjugation of laminin peptides………………....19
2.4.4. Dose-dependent cell adhesion activity on A99-ELP-R coated plates…….20
2.4.5. Cell attachment and spreading activity on A99-ELP coated plates……….22
2.4.6. Visualizing cell spreading on A99-ELP-R coated plates……………….…24
2.5. Discussion………………………………………………………………………...…26
2.6. Summary…………………………………………………………………………….32
2.7. Acknowledgments…………………………………………………………………...32
CHAPTER 3: LAMININ-DERVIVED ELPs OPTIMIZE CELL SPREADING………….33
3.1. Abstract……………………………………………………………………….……..33
3.2. Introduction…………………………………………………………………….……33
3.3. Materials and Methods………………………………………………………………37
3.3.1. Construction, expression and purification of LELPs…………………...…37
3.3.2. Measuring the hydrodynamic radius of the LELPs……………………….42
3.3.3. Defining the lower critical solution temperature………………………….42
3.3.4. Cell culture………………………………………………………………...42
3.3.5. Determining the cell adhesion and spreading activity…………………….43
3.3.6. Evaluating various concentration ratios and their cell adhesion activity.…44
3.3.7. Inhibition assay……………………………………………………………44
3.3.8. Immunofluorescence staining and confocal microscopy………………….45
3.3.9. Cell adhesion and neurite outgrowth…………………………………...…46
3.3.10. Statistics……………………………………………………………….…46
3.4. Results…………………………………………………………….…………………47
3.4.1. Generation of a library of thermo-responsive biologically active laminin
peptide-ELPs……………………………………………………………….…….47
3.4.2. Protein purification and characterization………………………………….49
3.4.3. Cell adhesion activity of laminin peptide-ELP coated plates………….….54
3.4.4. Cell spreading and morphology on laminin peptide-ELP coated plates…..55
3.4.5. Cell adhesion activity with mixed laminin peptide-ELPs…………………59
viii

3.4.6. Inhibition of HDF adhesion and spreading………………………………..64
3.4.7. Neurite outgrowth of PC-12 cells on laminin peptide-ELP coated plates...65
3.5. Discussion………………………………………………………………………...…68
3.6. Summary…………………………………………………………………………….73
3.7. Acknowledgments………………………………………………………………...…73
CHAPTER 4: TEMPERATURE-RESPONSIVE ESTROGEN RECEPTOR FUSIONS
SELF-ASSEMBLE INTRACELLULARLY………………………………………………….74
4.1. Abstract…………………………………………………………………………...…74
4.2. Introduction………………………………………………………………………….74
4.3. Materials and Methods………………………………………………………………77
4.3.1. Plasmid construction………………………………………………………77
4.3.2. Cell culture and DNA plasmid transfection……………………………….78
4.3.3. Immunoblot detection……………………………………………………..79
4.3.4. Immunofluorescence and confocal microscopy………………...…………79
4.3.5. Subcellular localization………………………………………………...….80
4.2.7. Data and statistical analysis…………………………………………….....80
4.4. Results……………………………………………………………………………….81
4.4.1. Generation of thermally responsive ELP-ERα……………………………81
4.4.2. Temperature-triggered microdomain formation of ELP-ERα…….………83
4.4.3. Localization of puncta in the cytoplasm at greater temperatures………....84
4.5. Discussion…………………………………………………………….……………..86
4.6. Summary…………………………………………………………………………….87
4.7. Acknowledgments…………………………………………………………………...88
CHAPTER 5: CHARACTERIZATION OF FLOTILLIN-1 ELASTIN-LIKE
POLYPEPTIDE FUSIONS…………………………………………………………………….90
5.1. Abstract………………………………………………………………………...……90
5.2. Introduction………………………………………………………………………….91
5.3. Materials and methods……………………………………………………………....94
ix

5.3.1. Construction of FLOT1 plasmids…………………………………………94
5.3.2. Cell culture………………………………………………………………...97
5.3.3. Stable cell line generation…………………………………………………97
5.3.4. Immunoblot analysis………………………………………………………99
5.3.5. Immunofluorescence and confocal microscopy…………………………...99
5.3.6. Live cell imaging and determination of the transition temperature……….99
5.3.7. Tracking internalization and colocalization……………………………...100
5.3.8. Statistics………………………………………………………………….101
5.4. Results……………………………………………………………………………...101
5.4.1. Generation of a small library of FLOT1-ELPs………………………..…101
5.4.2. Successful generation of stable cell lines………………………………...102
5.4.3. Determination of the transition temperature……………………………..103
5.4.4. Cholera toxin B colocalizes with F1-V96 and is sent to the lysosome…..104
5.5 Discussion…………………………………………………………………………..106
5.6. Summary…………………………………………………………………………...108
CHAPTER 6: SUMMARY, CHALLENGES AND FUTURE PERSPECTIVES……...…108
6.1. Summary…………………………………………………………………………………...108
6.2. Challenges………………………………………………………………………………….109
6.3. Future perspectives………………………………………………………………………...110
BIBLIOGRAPHY…………………………………………………………………...…...……111

x

LIST OF TABLES
Table 1. Summary of the amino acid sequences used for Chapter 2……………………………29
Table 2. Cassette of oligonucleotide sequences of the bioactive laminin peptides……………...38
Table 3. Recombinant laminin-derived elastin-like polypeptides evaluated in Chapter 3……....39
Table 4. Estimated purification yield and purity based on SDS-PAGE………………………....41
Table 5. Biological activity of LELPs……………………………………………...……………65
Table 6. Amino acid sequence of the plasmids for Chapter 4…………………………………...82
Table 7. Amino acid sequence of FLOT1 constructs……………...…………………………….95
Table 8. Summary of the fusion plasmids constructed for Chapter 5…………………………...98

xi

LIST OF FIGURES
Figure 1. Construction and expression of recombinant elastin-like polypeptide (ELP) with
laminin α1 chain A99 sequence peptide…………………………………………………………16
Figure 2. Oligonucleotide primer pair sequences used for the In-Fusion cloning reaction……..16
Figure 3. Detection and determination of the lower critical solution temperature……………...18
Figure 4. 100 µM of NHS-fluorescein conjugated A99-ELP-R incubated above the Tt………..19
Figure 5. Synthesis of o-chloroacetyl-N-hydroxysuccinimide………………………………….20
Figure 6. Conjugation of the laminin α1 chain peptide to I30 via chloroacetylation…………...20
Figure 7. Dose-dependent cell adhesion on A99-ELP-R coated plates………………………....21
Figure 8. Cell adhesion and morphology on various coated plates……………………………..23
Figure 9. Cell spreading activity………………………………………………………………...25
Figure 10. Schematic illustration of the strategy for engineering laminin bioactive peptide and
ELPs as a biomaterial to promote cell attachment and spreading……………………………….28
Figure 11. Average area of HDFs at various coating concentrations of A99-ELP-R at 4 ֯C or 70 ֯
C………………………………………………………………………………………………….31
Figure 12. Construction of plasmid to express LELPs………………………………………….48
Figure 13. Identity and phase behavior for LELP fusions………………………………………50
Figure 14. MALDI-TOF results…………………………………………………………………51
Figure 15. Hydrodynamic radius (Rh) of the LELPs……………………………………………52
xii

Figure 16. Measured transition temperature of the laminin peptide-ELPs……………………...53
Figure 17. Dose-dependent cell adhesion of LELP coated plates……………………………….54
Figure 18. Dose-dependent cell spreading on LELP coated plates……………………………...56
Figure 19. LELP fusions demonstrate distinct cell adhesion and morphology…………………58
Figure 20: Vinculin and F-actin arrangement during cell attachment and spreading…………...59
Figure 21. Cell attachment activity of mixed ratios……………………………………………..61
Figure 22: Synergistic cell adhesion and spreading activity of A99:A2G80-ELP……………...63
Figure 23. Brightfield micrograph images of 2x10
4
HDFs plated on LELP coated plates with
inhibitor…………………………………………………………………………………...……...66
Figure 24. Neurite outgrowth of PC-12 cell coated on LELPs………………………………….67
Figure 25. Purified I30 only and EF1m-ELP in Tm microcuvettes above Tt…………………...71
Figure 26. Design and illustration of proposed ELP-ERα assembly and confirmation of
expression……………………………………………………………………………………..…83
Figure 27. Temperature-triggered phase transition of ELP-ERα fusions…………………….…84
Figure 28. Microdomain formation is found more often in the cytoplasm than the nucleus…....86
Figure 29. Visual illustration and confirmation of FLOT1-ELPs……………………………...102
Figure 30. Cell staining pattern of FLOT1-ELPs represents different behaviors……………...104
Figure 31. Cholera toxin subunit B is internalized and colocalizes with F1-V96……………..105
xiii

ABBREVIATIONS
A2G80: Laminin α2 chain peptide (VQLRNGFPYFSY)
A99: Laminin α1 chain peptide (AGTFALRGDNPQG)
A99-ELP-R: Recombinant A99 fused to I30
A99-ELP-C: Chemically conjugated CGG-A99 containing peptide to I30
AG73: Laminin α1 chain peptide (RKRLQVQLSIRT)
BSA: Bovine serum albumin
CS: Charcoal-stripped
CTxB: Cholera toxin subunit B
E2: Estradiol
ECM: Extracellular matrix
EDTA: Ethylenediaminetetraacetic acid
EF1m: Laminin α1 chain peptide (LQLQEGRLHFMFD)
ELP: Elastin-like polypeptide
ERα: Estrogen receptor α
F1: Wild type flotillin-1 fused to EGFP
F1-V72: Thermally responsive flotillin-1 fused to EGFP and (VPGVG)72
F1-V96: Constitutively ‘on’ flotillin-1 fused to EGFP and (VPGVG)96
xiv

F1-A96: Temperature-insensitive flotillin-1 fused to EGFP and (VPGAG)96
FOV: Field of view
I30: Elastin-like polypeptide with a sequence of (VPGIG)30
IDP: Intrinsically disordered proteins
HDF: Human dermal fibroblast
LCST: Lower critical solution temperature
LELP: Laminin-derived elastin-like polypeptide
MW: Molecular weight
OD: Optical density
PBS: Phosphate-buffered saline
PRe-RDL: recursive directional ligation by plasmid reconstruction
ROI: region of interest
RT: Room temperature
Tt: Transition temperature
V96-ERα: V96-ELP fused to the N-terminus of full length ERα

xv

ABSTRACT
This dissertation harnesses the power of synthetic biology and protein engineering to generate
peptide and protein fusions that strategically employs stimuli-responsive biopolymers called
elastin-like polypeptides (ELPs). ELPs are a class of biopolymers that undergo a reversible phase
separation in response to heat where they self-assemble to form an insoluble coacervate. This
unique characteristic, when coupled to a domain or protein can drive biomolecules to concentrate
and can modify the properties or behavior of fusion. The bioactivity of these complexes are
explored in this dissertation where these fusions with multi-functional properties are characterized.
In Chapter 1, a proof-of-concept using a short hydrophobic ELP (I30) with a biologically active
laminin peptide (A99) from the extracellular matrix is produced. The fusion is compared against
various conditions and demonstrate to have favorable biophysical properties for purification and
bioactivity in cell adhesion and spreading. Chapter 2 expands on this technology and introduces a
library of laminin peptides (A2G80, AG73 and EF1m) from different domains of laminin fused to
the ELP. Their biological activity in the context of cell attachment and spreading is assessed and
shown to have synergy when mixed. Additionally, they promote neurite outgrowth of neuronal
cells. Chapter 3 moves into the cell and studies how classic nuclear hormone receptor, estrogen
receptor α (ERα) behaves when fused to an ELP. At temperatures below 23 C, ELP-ERα fusions
remain soluble both in the cytoplasm and the nucleus; however, with the addition of estradiol
promotes concentration and microdomain formation which is found predominantly in the
cytoplasm. Finally, in Chapter 4, a less understood multi-functional protein called flotillin-1 is
fused to a library of ELPs and are briefly characterized. Flotillin-ELP fusions mobilize rapidly
throughout the cell and shuttle between the cytoplasm and plasma membrane. These chapters
demonstrate how ELPs unique characteristics may modulate biologically relevant pathways.

CHAPTER 1: INTRODUCTION
1.1. Cell Signaling
Cell communication, signaling or signal transduction is a transmission method cells use to
‘send messages’ to regulate all biological processes within the human body. Similar to modern day
society, signaling involves whole body of networks and has a lot of moving parts to control specific
cell functions.
There are two main classes of transmission: extracellular signaling and intracellular
signaling. Most of the communication within the human body is mediated via extracellular
signaling where messages, coming from neighboring cells or from distant locations, modulate the
cells’ behavior in a perplexing, yet specific manner. This complexity, owing to a variety of
signaling molecules such as chemical messengers, proteins or lipids and its many binding partners,
are highly regulated processes with significant ‘checks and balances’ (feedback-loops) to ensure
the messages are appropriately received and transcribed. The messages are usually received
through cell surface receptors (such as receptor tyrosine kinases, g protein-coupled receptors or
other transmembrane receptors) which in turn, activate a cascade of intracellular signaling
pathways- a process that occurs within the cell that sorts the signals and sends the message to its
final destination to regulate the cell. However, in a few cases, some hydrophobic signals such as
steroid hormones can passively diffuse through the plasma membrane and generate intracellular
signals that alters the behavior of the cell.
Cell communication ideally should respond rapidly to messages; however, this is not
always the case. The speed of signaling may be broken down into two categories: fast or slow.
Fast signaling (in the order of seconds to minutes) occurs when a specific response only changes
2

what is already present in the cell such as phosphorylation of a protein or activation of an ion-
gated channel. Slow responses require much more time (in the order of minutes to hours) because
their biological response usually require changes in gene expression and de-novo synthesis of
proteins. A few reasons for these differences are to save on the cell’s raw material and energy, and
also to prevent having extra proteins available that are typically tightly regulated. Interestingly, in
addition to the differences in the communication and response speed, various cell types can also
respond differently to the same signaling molecule. An example of this are catecholamines which
are messages that control our ‘fight or flight’ responses. When catecholamines such as epinephrine
bind to cardiomyocytes, this activates the β1-receptors, increasing cardiac output by increasing the
heart rate and contractile force. However, the same ‘messenger’ when bound to β2-receptors found
at the bronchioles, cause smooth muscle relaxation and therefore enables more respiration. These
examples demonstrate the importance of the signaling molecule, its target and the cell type, and
how these differences can have major distinctions in the cell’s behavior.
Cell communication is a highly orchestrated and precise process, and much effort has been
invested into understanding individual pathways and their relationship to changes in the cell’s
behavior. This intense area of study has been expanded into biomedical research and many
scientists have focused on engineering all aspects of these messages to regulate the behavior of the
cell.
1.2. Thermo-responsive biopolymers
Elastin-like polypeptides (ELPs) are synthetic biopolymers that self-assemble in response
to heat. These biopolymers are inspired by the hydrophobic domain of human tropo-elastin found
in the extracellular matrix and have advantageous biophysical characteristics suitable for a variety
of in-vitro and in-vivo applications. ELPs consist of a pentameric amino acid sequence of
3

(VPGXaaG)n where Xaa represents any guest amino acid and n is the number of repeating units.
They undergo a reversible phase transition at the lower critical solution temperature (LCST) also
known as the transition temperature (Tt). When below the Tt, ELPs remain soluble, yet, when
heated to above the Tt, ELPs coalesce, forming an insoluble, polymer rich coacervate. The Tt is
tunable and is influenced by the guest amino acid and the pentapeptide length. Since they are
recombinantly synthesized, this enables precise control over these parameters, in addition to the
inclusion of any artificial gene or peptide directly into its sequence. This allows for ELPs to be
incorporated into functional domains or whole proteins to develop novel fusions with multi-
functional properties. One major advantage of ELPs is that they can be purified in large quantities
from E. coli by exploiting their thermal responsiveness. This ELP-mediated phase separation does
not require laborious or expensive chromatography and is achieved by cycling the temperature
followed by centrifugation to remove any contaminants.
ELPs are becoming an increasingly popular class of biomolecules that have gradually been
applied to generate various peptide or protein fusions for a diverse number of biomedical
applications such as tissue engineering, drug delivery or even as molecular switches (Pastuszka,
Okamoto et al. 2014, Ryu and Raucher 2015, Doberenz, Zeng et al. 2020). They are especially
attractive for tissue engineering due to their biophysical properties and biocompatibility. Cell-
recognizing adhesion peptides and crosslinking amino acid moieties can be introduced into the
sequence to regulate the elasticity and mechanical properties as hydrogels to mimic the body
natural elements (Cai, Dinh et al. 2014). In addition to the ELPs physical properties, their phase
behavior is an incredibly useful attribute for regenerative medicine. For example, especially
designed healing ELP fusions can be introduced as a soluble solution below the Tt as a minimally
invasive injection and form a gel at the affected area when acclimated to body temperature
4

(Kimmerling, Furman et al. 2015). ELPs have also been extensively studied as a drug delivery
vehicle because of their biophysical properties and adaptability. The pentapeptide amino acid
sequence can be designed to address the challenges of drug delivery. Improvements in
pharmacokinetic parameters such as the half-life or its bioavailability are achievable with ELPs
(Despanie, Dhandhukia et al. 2016). Additionally, they can also promote targeted approaches to
reduce unwanted cell cytotoxicity. An example of this accomplishment was to fuse a specific
recognition site to one end of the ELP, while carrying the payload on the other end (Guo, Lee et
al. 2018). ELPs have been used to help increase the solubility of drugs and also have formed
encapsulated drug nanoparticles as well. Most recently, ELPs have been investigated inside living
cells to act as molecular switches and control vital biological processes (Pastuszka, Okamoto et al.
2014, Li, Tyrpak et al. 2018). ELP fused to full length proteins have formed microdomains upon
heat stimulation both at the cell surface and in the cell to modulate their respective pathways. This
is particularly useful because the process is rapid and reversible, whereas other methods to control
proteins are slow, irreversible or have off-target side effects.
Thermally responsive ELP behavior is a valuable characteristic that can be applied for a
variety of biomedical applications. The biopolymers are genetically encoded and enables full
customization of their sequence to accommodate any engineering requirements.

5

CHAPTER 2: EXTRACELLULAR MATRIX MIMETICS TO
PROMOTE CELL ADHESION AND ATTACHMENT
1

2.1. Abstract
The extracellular matrix (ECM) is comprised of a large network of proteins that are essential for
tissue development and repair. A bioactive RGD-containing peptide from laminin α1 chain, A99
(AGTFALRGDNPQG), promotes strong cell attachment and has demonstrated utility in cell
culture and tissue engineering. Various materials can be utilized as a scaffold for bioactive
peptides; however, it may be advantageous to design materials that use bioconjugation strategies
that do not affect bioactivity, generate homogenous products, and can be produced at scale. This
report is the first to compare the methods for preparing chemically conjugated and recombinant
A99 to elastin-like polypeptides (ELPs) as the scaffold and characterize the biological and cell
attachment activity using human dermal fibroblasts (HDFs). ELPs are biocompatible protein-
polymers that are also thermo-responsive. Below a lower critical solution temperature (LCST),
they are highly soluble. Above the LCST, ELPs phase separate into a polymer-rich liquid, known
as a coacervate. Both chemically conjugated and recombinant fusion between A99 and an ELP
(A99-ELP-R) show dose-dependent cell attachment. In addition, coating above the LCST provides
better cell spreading compared to coating at 4°C. ELPs provide an excellent structural framework
for deposition of bioactive peptides of the ECM, and their intrinsic biophysical properties make
laminin peptide-ELPs promising biomaterials for cell culture and tissue engineering.
1
This chapter was referenced to a manuscript, titled “Evaluation of extracellular matrix mimetic laminin
bioactive peptide and elastin-like polypeptide” by Truong et al.
6

2.2. Introduction
Tissue regeneration such as wound healing and revascularization, includes a variety of
physiological processes involving cell migration, adhesion, differentiation and proliferation
(Frantz, Stewart et al. 2010, Lanza, Langer et al. 2014). These processes are promoted by proper
stimulation from the extracellular matrix (ECM), which provides the mechanical structure, growth
factors and recognition motif for growth and development (Olczyk, Mencner et al. 2014, Akter
2016). Following an insult or injury, it is crucial to restore the function of damaged tissues. Since
these processes play a key role in tissue regeneration, synthetic biomaterials have been engineered
to emulate the native ECM. Therefore, ECM mimetics have become very attractive for
regenerative medicine applications and have potential to reduce the prevalence of chronic wounds
(Ho, Walsh et al. 2017).
Major components found at the ECM include collagen, fibronectin and laminin. While they
all have important biological functions, laminin considerably contributes to tissue development
and regeneration. Laminins are large heterotrimeric glycoproteins that consist of an α, β and γ
chain and are most well-known for their cell adhesion activity. They interact with a variety of cell
surface receptors such as integrins and syndecans. Laminin dysregulation contributes to many
epithelial disorders (McGowan and Marinkovich 2000). Currently, 19 isoforms of laminin have
been identified, which consist of combinations of five α-chains, three β-chains, and three γ-chains.
These isoforms have variable expression within the body that are not only based on location, but
also the developmental stage and external stimulating factors (Tunggal, Smyth et al. 2000, Durbeej
2010). Since they promote diverse biologically relevant activities, including cell migration, neurite
outgrowth, differentiation, and tumor angiogenesis and metastasis, they are a promising
7

therapeutic target for tissue repair (Kalluri 2003, Sasaki, Fassler et al. 2004, Yamada, Mori et al.
2016).
Several bioactive peptides from mouse laminin α1 chain were previously identified by
screening the full-length protein using a library of synthetic peptides that span its entire amino acid
sequence. Small peptides approximately 12 amino acids in length were synthesized, where each
peptide consisted of an overlap with four amino acids with the preceding peptide (Nomizu,
Kuratomi et al. 1998). All peptides were screened for cell adhesion activity, and cellular
morphology was characterized. A table of bioactive laminin peptides were identified (Nomizu,
Kuratomi et al. 1998, Malinda, Nomizu et al. 1999, Mochizuki, Kadoya et al. 2003). For example,
AG73 (RKRLQVQLSIRT) binds syndecans and promotes cell adhesion with membrane ruffling.
A99 (AGTFALRGDNPQG) and EF1 (DYATLQLQEGRLHFMFDLG) interact with integrin
αvβ3 and α2β1, respectively, which promotes cell spreading with focal adhesions. A99 was
selected over EF1 as the candidate peptide for this study because it contains the canonical RGD
motif, its impact on the morphology of cell spreading can be easily evaluated, and it has good
aqueous solubility.
Biomaterials that are used as a matrix for tissue repair and healing should mimic the native
ECM microenvironment to support the necessary cellular responses needed to improve the rate
and success of tissue regeneration. Historically, synthetic and naturally occurring polymers such
as poly(N-isopropylacrylamide), poly(vinyl alcohol), alginate, gelatin, hyaluronan or chitosan
have been explored as scaffolds, but these polymers alone lack the necessary factors to promote
cell attachment and growth (Zhu and Marchant 2011, Fujimori, Kumai et al. 2017). This weakness
led to biochemical approaches, crosslinking bioactive peptides or other factors to help mimic the
ECM. Furthermore, these bioactive peptides found their way into recombinantly expressed E. coli
8

as an alternative approach to sometimes harsh bioconjugation strategies (Meyer and Chilkoti 1999,
Nettles, Chilkoti et al. 2010, Kakinoki, Nakayama et al. 2014). Specifically, these bioactive
peptides have been carefully engineered to be incorporated with biopolymers that act as scaffolds,
which enhance the biophysiochemical properties or production of these small peptides.
Elastin-like polypeptides (ELPs) are thermo-responsive biopolymers derived from a
structural motif in human tropoelastin, a component of connective tissue itself. ELPs are attractive
in tissue engineering applications because they are genetically encoded and can be synthesized
with high fidelity with respect to their amino acid sequence, molecular weight, and polydispersity.
In addition, they are biocompatible, biodegradable and can be produced in large yields through
bioprocess engineering. The ELPs evaluated here consist of a pentameric amino acid repeat (Val-
Pro-Gly-Xaa-Gly)n, where Xaa can be any guest amino acid residue and n is the number of repeats.
ELPs undergo an inverse phase transition, where below a lower critical solution temperature
(LCST), ELPs are highly water-soluble. When the temperature is raised above this LCST, also
known as the transition temperature (Tt), they phase separate into a secondary aqueous phase called
a coacervate. This phenomenon is due to their amino acid sequence composition and
hydrophobicity, where they undergo a conformational change from random coils to type II β-turn
spirals, thus promoting this phase transition. This process is rapid and reversible and has been used
as a method for protein purification by mediated phase transition. These unique properties have
made this biomaterial increasingly popular as a scaffold for tissue and cell engineering (Mochizuki,
Yamagata et al. 2007, Iorio, Troughton et al. 2015, Akter 2016, Zhu, Cankova et al. 2018).
This study introduces a new bioactive laminin peptide and ELP construct, A99
(AGTFALRGDNPQG) conjugated to I30 (VPGVG)30 by both molecular cloning and chemical
9

conjugation. This study demonstrates the biological activity and usefulness of laminin peptide-
ELPs as a biomaterial.

2.3. Material and methods
2.3.1. Reagents
Reagents. N,N-dimethylformamide (DMF, 10344-76) and tetrahydrofuran (THF, 40060-00) was
purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Hexane (17921-04) was purchased from
Nacalai Tesque, Inc. (Kyoto, Japan). Chloroacetic acid (033-02232) was purchased from Wako
Pure Chemical Ind., Ltd (Tokyo, Japan). N-hydroxy succinimide (NHS, A00013) and N,N’-
dicycloheylcarbodiimide (DCC, A00005) was purchased from Watanabe Chemical Ind., Ltd
(Hiroshima, Japan). 5/6-carboxyfluorescein succinimidyl ester (NHS-fluorescein, 46410) was
purchased from ThermoFisher Scientific (Waltham, MA).
2.3.2. Construction, expression and purification of ELPs
I30 was generated in a pet25b(+) bacterial vector using recursive directional ligation as previously
described (McDaniel, Mackay et al. 2010, Janib, Pastuszka et al. 2014). The A99 peptide sequence
(AGTFALRGDGNPQ) was queried with BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to
obtain the original nucleotide sequence corresponding to mouse laminin α1 chain and the In-
Fusion Cloning method (Takara Bio USA, Mountain View, CA) was selected for rapid cloning.
Briefly, 15 base pairs before and after the NdeI restriction cut site found immediately at the start
codon of the ELP gene was flanked between the A99 nucleotide translating sequence for
homologous, in-frame recombination. In addition, a single glycine amino acid was added as a
spacer between A99 and I30. The oligonucleotides were ordered (ThermoFisher Scientific,
10

Waltham, MA), and cloning was performed using the manufactures suggested protocol. Successful
cloning of the complete sequence notated as, A99-ELP-R, was verified by Sanger sequencing
(Genewiz, Tokyo, Japan).
The plasmid DNA was first amplified in DH5α competent cells, then transformed in BL21
(DE3) competent cells (ThermoFisher Scientific, Waltham, MA) for subsequent protein
expression. ELPs were purified using induction of ELP-mediated phase separation.(Meyer and
Chilkoti 1999) In brief, transformed E. coli was grown in terrific broth with constant agitation at
37 ֯C overnight for approximately 16 h. The following day, the medium was removed by
centrifugation (6000 g) and resuspended in ice-cold phosphate buffered saline (PBS). Cells were
lysed by sonication and excess debris and nucleic acids were removed using poly-ethyleneimine
(PEI, 408700-250ML, Sigma-Aldrich, St. Louis, MO) on a rotator (RT-50, Taitec, Saitama, Japan)
for 10 min at 4 ֯C and centrifugation (3000 g) at 4 ֯C. ELP coacervation was triggered by the
addition of 1M sodium chloride and heating at 37 ֯C, which was followed by centrifugation (3000
g) for 10 min at 37 ֯C (hot spin). The supernatant was discarded and ELP pellets were then carefully
resolubilized on ice in cold PBS. The solution was centrifuged (3000 g) at 4 ֯C for 10 min (cold
spin), and the supernatant was collected into a new tube, while the debris were discarded. Hot and
cold spins were performed over 3 rounds in which the last round was resolubilized in cold MilliQ
water to obtain pure ELPs above 95% (Despanie, Dhandhukia et al. 2016). ELPs were frozen at -
80 ֯C and subsequently lyophilized. Aliquots were resuspended in PBS prior to use. The yield was
between 20 to 35 mg/L. Both I30 and A99-ELP-R protein purified products and molecular weights
were verified by SDS-page using a 4% stacking gel and 15% resolving gel, then stained briefly
with Coomassie Brilliant Blue. The expected amino acid sequence for I30 is MG(VPGIG)30Y,
which has an expected molecular weight (MW) of 12.9 kDa. The expected amino acid sequence
11

for A99-ELP-R is MAGTFALRGDNPQGGMG(VPGIG)30Y, which has an expected MW of 14.2
kDa (Wilkins, Gasteiger et al. 1999). A99-ELP-R was modified at the N-terminal primary amino
using NHS-fluorescein in DMF following conjugation at a molar excess of 15:1 for 2 h in PBS,
which was purified using ELP-mediated phase separation.
The ELP concentrations were determined by first diluting the ELPs 1:1 in 6 M guanidine
HCl to unfold any coacervates, then the optical density (OD) was measured at 280 nm with a 0.5
mm light path using a spectrophotometer (Multi-Skan Go, ThermoFisher, Scientific, Waltham,
MA). The Beer-Lambert law was applied to calculate the concentration:
𝐸𝐿𝑃 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑀 ) =
𝑂𝐷
280
× 𝑑𝑖𝑙 𝑢 𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 ε × l (cm)

where the estimated molar extinction coefficient, ε, of both ELPs are 1285 (M
-1
cm
-1
), which is
solely attributed to a carboxy-terminal tyrosine.
2.3.3. Determination of the lower critical solution temperature
A99-ELP-R thermal phase behavior was evaluated by monitoring the OD at 350 nm using a
temperature-control equipped spectrophotometer (DU/800 UV-vis, Beckman Coulter, CA). 50 µM
of A99-ELP-R was loaded into a cuvette and the Abs was measured as a function of temperature
(1 ֯C/min). The LCST is defined at the occurrence of the maximum first derivative of the optical
density at 350 nm. Afterwards, the transition temperature as a function of concentration was
plotted by utilizing the same method.
2.3.4. Peptide synthesis and conjugation
A99 or cysteine-containing A99 peptide was manually synthesized by using the 9-
fluorenylmethoxycarbonyl (Fmoc)-based solid phase method with a rink amide resin previously
12

described (Nomizu, Kuratomi et al. 1998). The N-terminus for the cysteine containing A99
includes two glycine amino acid residues as a spacer (CGG-A99, CGG-AGTFALRGDGNPQ).
The purity and accuracy of the peptides was confirmed using analytical HPLC and high-resolution
mass spectrometry. First, the synthesis of the peptide-polypeptide conjugate required an
intermediate step. Chloroacetylation of the N-terminus of I30 was performed with 1-2 mg of I30
dissolved in 0.1 M potassium phosphate pH 9. This was mixed with at least 15 mmol excess of N-
(chloroacetoxy)succinimide in 1 drop of DMF at 4 ֯C for 2 h. Purification of chloroacetyl-I30 was
performed by induction of ELP-mediated phase separation. This intermediate was resuspended in
300 µl of 0.1 M potassium phosphate and 15 mmol excess of CGG-A99 in 0.1 M potassium
phosphate was mixed, titrated to pH 8 and incubated on a tube rotator at 4 ֯C overnight. Unbound
CGG-A99 and salt was removed by 3 rounds of ELP-mediated purification and the conjugate was
resuspended in PBS. The products that are conjugated are designated as A99-ELP-C.
2.3.5. Cell culture
Human dermal fibroblasts (HDFs) (#106-05a, Cell Applications, Inc., San Diego, CA) were
maintained in DMEM (#C11885500BT, ThermoFisher Scientific, Waltham, MA) supplemented
with 10% FBS (#26140-079, ThermoFisher Scientific, Waltham, MA), 100 U/ml penicillin and
100 µg/ml streptomycin at 37 ֯C, in 5% CO2 humidified tissue culture incubators. Cell line passage
number was less than 23 for all experiments reported in this chapter.
2.3.6. Dose-dependent assay
Cell adhesion activity was assessed at various coating concentrations of A99-ELP-R. A99-ELP-R
(0.025, 0.1, 0.2, 0.5, 1, 10, 50 µM) was coated in 96-well non treated, flat bottom, polystyrene
plates (#1860-096, Iwaki, Shizuoka, Japan) in triplicate for 1 h at 70 ֯C (above the Tt at most
13

concentrations), then washed with PBS three times. Afterwards, 2x10
4
HDFs were plated in each
well with blocking media (DMEM, 0.1% BSA, without serum) and incubated at 37 ֯C for 1 h. The
cells were subsequently fixed with crystal violet for 5 min and washed with water. Brightfield
microscopy images (a minimum of 3 random images, per well in triplicates) were collected (BZ-
X810, Keyence, Osaka, Japan) using a chrome-free infinity, flat field optical, fluorite aberration
corrected, phase contrast 10x objective lens (numerical aperture of 0.30), and the number of
attached cells was counted.
2.3.7. Cell attachment and spreading assay
Cell attachment and spreading activity was assessed using the same methods as described above
in the dose-dependent assay, except 5x10
3
HDFs were plated. A99-MB-chitosan, which was
previously evaluated was added as a control (Mochizuki, Kadoya et al. 2003). Briefly, MB-
chitosan was dissolved in 4% Acetic acid (20 µg/ml) and 50 µl was added to 96-well plates and
allowed to dry overnight. 20 µl of 1 mM of CGG-A99 peptides were mixed in 50 µl of 200 nM
sodium phosphate and 20 µl of MilliQ H2O, then incubated at room temperature (RT, 24 ֯C)
overnight while protected from light. Brightfield microscopy images were collected and the area,
perimeter and maximum length was analyzed using CellProfiler (Carpenter, Jones et al. 2006).
Briefly, the pipeline was built as follows- images were imported into CellProfiler and metadata
extracted using folder name containing the date, experiment title, temperature, incubation time and
concentration. Images were converted to greyscale and inverted. Next, uneven illumination from
the microscope was corrected. Background noise was removed using a gaussian blur, and cells
were detected using a stricter threshold factor of 0.8. Objects detected at borders or that were either
too small or too large compared to a predefined value were masked from the analysis. The masked
portions were expanded from its original point and another gaussian blur was performed. Each
14

object was then redetected with a looser threshold of 0.6. After the detection of each object, each
image was visually inspected manually for accuracy of the region of interest (ROI) and corrected
as needed. CellProfiler reported the data in pixel value, which was converted to µm (132 pixels/100
µm).
2.3.8. Immunofluorescence staining and fluorescence microscopy
1 µM of A99-ELP-R was coated onto 96-well non-treated, flat bottom, polystyrene plates as used
previously for 1 h at 70 ֯C. The solution was then removed, and plates were washed with RT PBS
three times. 5x10
3
HDFs in blocking media (1% BSA in DMEM) were seeded into each well in
triplicate and allowed to incubate at 37 ֯C in 5% CO2 humidified incubators for 2 h. After
incubation, the cells were fixed directly onto the 96-well plate wells with 4% paraformaldehyde
(PFA) for 15 min. PFA was quenched with 0.1 M glycine in PBS (pH 7.5) for an additional 20
min. Plates were washed with PBS 3 times (5 min each). The cells were permeabilized with 0.5%
Triton X-100 (T-9284, Sigma-Aldrich, St. Louis, MO) for 10 min, washed with PBS 3 times (5
min each), and blocked with 1% BSA in PBS for 30 min. 1:400 of mouse anti-vinculin antibody
(V9131, Sigma-Aldrich, St. Louis, MO) was incubated for 1 h at RT (24 ֯C) and washed with PBS
3 times. Thereafter, 1:100 goat anti-mouse IgG Alexa Fluor 488 secondary antibody and 1:200
Alexa Fluor 594 phalloidin conjugated probe (A11029 and A12381, respectively, ThermoFisher
Scientific, Waltham, MA) were incubated for an additional 1 h at RT. The media was replaced
with 1:1000 DAPI (D3571, Invitrogen, Paisley, PA, UK) in PBS, and plates were incubated at RT
for 5 min and washed with PBS 5 times. All the liquid was carefully removed and a layer of para
phenylenediamine (PPD) anti-fade reagent (0.1% w/v PPD in 10% PBS and glycerol) was added
to cover the entire cell surface. Plates were covered with aluminum foil and stored at 4 ֯C until
imaging. Fluorescent images were collected using the same BZ-X810 microscope with a chrome-
15

free infinity-corrected, flat field optical, fluorite aberration corrected 20x objective lens (numerical
aperture of 0.45).
2.3.9. Statistics
All experiments were replicated at least 3 times and the data presented as Mean ± SD or CI. All
statistical analysis was performed using GraphPad Prism 6 (La Jolla, California,
http://www.graphpad.com). For each analysis (area, perimeter and major axis length), a one-way
ANOVA comparing each group was executed followed by Tukey’s post-hoc test. A p-value of
0.05 or less was considered to be statistically significantly different.

2.4. Results
2.4.1. Generation of thermo-responsive bioactive laminin peptide-ELPs
Fusion of the laminin peptide sequence A99 to I30 was generated using the In-Fusion cloning
method, which was selected for its robustness, ease-of-use and one step cloning, compared to the
multi-step process of DNA restriction and ligation. Figure 1A) provides the amino acid sequence
from the N-terminus starting with A99, including the linker/spacer and the partial beginning of the
I30 sequence (SnapGene®, GSL Biotech). Complete details of the oligonucleotides can be found
in Figure 2. After successful homologous recombination of the oligonucleotide primers and
plasmid, diagnostic restriction digest of A99-ELP-R showed a similar base pair size as compared
to I30 alone. Figure 1B) provides an image of the plasmid map. The reconstructed plasmid
insertion sequence and appropriate reading frame was verified by Sanger sequencing.

16

Figure 1. Construction and expression of recombinant elastin-like polypeptide (ELP) with laminin
α1 chain A99 sequence peptide. A) Nucleotide and protein sequence map. A99 (AGTFALRGDNPQG)
was fused to the amino terminus of I30 using In-fusion Cloning at the NdeI restriction site of previously
constructed pet25b I30 (only partial of I30 is shown). ELP consist of a pentameric repeat of (Val-Pro-Gly-
Ile-Gly) 30 as shown in the black box. B) Complete plasmid map of recombinantly fused A99-ELP-R in the
pet25b(+) vector, where the A99 sequence is at the N-terminus and I30 sequence is at the C-terminus. C)
SDS-page of purified I30 (12.9 kDa) and A99-ELP-R (14.2 kDa) after 3 rounds of ITC, stained with
Coomassie Brilliant Blue. SDS-page image was modified by switching the color image to greyscale and
increasing the contrast.

Figure 2. Oligonucleotide primer pair sequences used for the In-Fusion cloning reaction. Both ends
flanking the A99 sequence matches 15 or 18 base pairs N to C terminal end respectively for homologous
recombination for the insertion of A99.

17

2.4.2. Protein purification and characterization
I30 and A99-ELP-R were prepared by recombinant protein production in E. coli and purified by
ELP-mediated phase separation. The molecular weight and purity of I30 and A99-ELP-R were
verified by SDS-PAGE and Coomassie staining. I30 and A99-ELP-R’s expected molecular
weights were 12.9 kDa and 14.2 kDa, respectively, which is consistent with the SDS-page results
(Figure 1C). Phase separation of A99-ELP-R was demonstrated in glass tubes by incubating 100
µM of fluorescein-conjugated A99-ELP-R in PBS in glass tubes below and above the expected Tt.
Below the Tt, the yellow transparent liquid indicates the ELPs are in solution, while above the T t,
coacervates form creating an opaque suspension (Figure 3A). Incubation of 100 µM coated A99-
ELP-R above the Tt on 96-well plates demonstrates the deposition of a visibly thin coat compared
to incubation below the Tt (Figure 3B), and distinct coacervates are observed on the plate (Figure
4). Exact measurement of the Tt was then performed by measuring the optical density (OD 350
nm) as a function of temperature (1 ֯C/min). The Tt is defined where the maximum first derivative
was observed (PBS alone was used as a negative control), whereby a 50 µM solution of A99-ELP-
R has a Tt of 24 ֯C (Figure 3C). Different concentrations of A99-ELP-R (3.13, 6.25, 12.5, 25 and
50 µM) were also assayed for Tt. These were plotted on a logarithmic scale, which reveals a log-
linear relationship between temperature and concentration (Figure 3D).

18

Figure 3. Detection and determination of the lower critical solution temperature. A) Recombinant
laminin α1 chain, A99 peptide fused to I30 (A99-ELP-R, 100 µM), was conjugated with fluorescein and
demonstrates temperature dependent reversible coacervation as depicted with the opaque color above the
LCST. B) Brightfield color image of 100 µM of A99-ELP-R coated for 1 h in 96-well plate at 4 or 37 ֯C.
The high concentration is used here as an example to visually demonstrate a thin layer that coats the wells.
C) The optical density was measured as a function of temperature (1 ֯C/min) for 50 µM A99-ELP-R and
PBS as a control. The transition temperature (24 ֯C) is defined as the maximum first derivative. D)
Transition temperature as a function of concentration for A99-ELP-R shows the expected concentration to
achieve coacervation. Dotted lines indicate the 95% confidence interval around the best-fit line.
19

Figure 4. 100 µM of NHS-fluorescein conjugated A99-ELP-R incubated above the Tt. Fluorescent
images demonstrate coacervates when 100 µM of fluorescein conjugated A99-ELP-R is incubated at 37 ֯C
for 1 h which adhere to non-TC treated 96-well plates. Labeling efficiency of NHS-fluorescein to A99-
ELP-R is approximately 8.4%.

2.4.3. Chemical synthesis and conjugation of the laminin peptides
Traditional Fmoc solid phase peptide synthesis was used to prepare A99 and cysteine-containing
A99 (CGG-A99). Their purity and molecular mass were determined by reverse phase HPLC and
high-resolution mass spectrometry respectively. The N-terminal domain of I30 was
chloroacetylated using N-(chloroacetoxy)succinimide, then CGG-A99 was conjugated under basic
conditions at 4 ֯C overnight (synthesis of N-(chloroacetoxy)succinimide is found in Figure 5). This
reaction yielded 50% (for 2 steps) of chemically conjugated A99-ELP-C (complete schematic
found in Figure 6). Unbound CGG-A99 was removed by 3 rounds of ELP-mediated purification.

20

Figure 5. Synthesis of o-chloroacetyl-N-hydroxysuccinimide. This was synthesized by dissolving DCC
(3.1 g, 15 mmol) in a minimal amount of THF and added dropwise to a solution that was cooled to 0 ֯C of
NHS (1.74 g, 15 mmol) and chloroacetic acid (1.42 g, 15 mmol) in THF (total 200 ml). The reaction was
stirred at 0 ֯C for 5 h, the precipitates filtered, and the filtrate was washed with 10mL THF. 10 ml of hexane
was added and allowed to sit at 5 ֯C for 12 h. The precipitates filtered again, and filtrate concentrated in
vacuo. 100 ml of hexane was added and set aside at 5 ֯C for 2 h and subsequently filtered. 2.87 g of white
powder (98%) was collected and verified by H NMR (CDCl 3) δ 4.36 (s, 2H); 2.86 (s, 4H).

Figure 6. Conjugation of the laminin α1 chain peptide to I30 via chloroacetylation. The N terminus of
I30 was first conjugated with N-(chloroacetoxy)succinimide for the next coupling reaction. Cysteine
containing laminin α1 peptide A99 (CGG-AGTFALRGDNPQG) was conjugated by the reactive
chloroacetyl group. This 2-step reaction provided a 50% yield.

2.4.4. Dose-dependent cell adhesion activity on A99-ELP-R coated plates
The cell attachment activity of A99-ELP-R was determined at various coating concentrations
(0.025, 0.1, 0.2, 0.5, 1, 10 50 µM). When A99-ELP-R was coated at 70 ֯C for 1 h, HDFs have poor
attachment activity below 0.5 µM of A99-ELP-R where less than 100 cells were identified per
field of view (FOV: 1449 x 1091 µm) (Figure 7A). On the other hand, 0.5 µM coating significantly
21

increases cell attachment to a mean of 490 cells per FOV (p ≤ 0.0001). At higher coating
concentrations of 1, 10 or 50 µM, maximal cell adhesion reached a plateau at a mean of 532, 550
and 513 cells per FOV respectively. Statistical analysis indicates that there is no significant
difference in attachment activity between 0.5 and 50 µM of A99-ELP-R (p = 0.2677). Microscopic
images also demonstrate very minimal spreading at 0.2 µM coating (or below) compared to 0.5
µM (Figure 7B).

Figure 7. Dose-dependent cell adhesion on A99-ELP-R coated plates. A99-ELP-R was coated in a 96-
well plate for 1 h at 70 ֯C and then 2x10
4
HDFs were seeded and incubated for 1 h at 37 ֯C. A) The average
number of attached cells per field of view (1449 x 1091 µm) was determined at various concentrations of
A99-ELP-R (0.025, 0.1, 0.2, 0.5, 1, 10, 50 µM). B) Representative microscopic images of the cells at two
coating concentrations (200 and 500 nM), between which there is a statistically significant difference in
cell adhesion activity (p ≤ 0.0001). Images were converted from color to grayscale. Error bars indicate
standard deviation.
22

2.4.5. Cell attachment and spreading activity on A99-ELP coated plates
HDF attachment and morphology were analyzed at different coating conditions (Figure 8).
Microscopic images demonstrate that when A99 peptide is coated without any scaffold, HDFs
have extremely poor attachment activity and the surfaces do not promote cell spreading. When 1
µM of I30 is coated at 70 ֯C, a few HDFs seem to non-specifically attach with minimal spreading.
A99-chitosan was previously shown to have excellent cell attachment and spreading activity and
was used here as a morphological control for spreading. When coated at 4 ֯C, A99-ELP-R showed
HDF attachment/adhesion and spreading, although less than compared to A99-chitosan, A99-ELP-
C and A99-ELP-R when coated at 70 ֯C. 1 µM of A99-ELP-C and A99-ELP-R, when coated at 70
֯C and incubated for 1 h at 37 ֯C both demonstrate excellent, yet similar cell attachment and
spreading activity.
Approximately 18,000 individual HDFs were analyzed among all the groups and
concentrations and the spreading activity was determined by measuring the area, perimeter and
major axis length of each cell (performed as triplicates, three times) (Figure 9A, B, and C). A99
peptide alone, when coated had minimal values among all of these categories (at 0.5 µM coating,
HDFs had a mean area of 642 µm
2
, major axis length of 31.03 µm, and perimeter of 109.8 µm).
Starting at 0.5 µM, which was demonstrated to be the minimal effective concentration based on
Figure 4, A99-ELP-R and A99-ELP-C coated at 70 ֯C led to significantly greater cell area (p = ≤
0.0001) and major axis length (p = ≤ 0.0001), compared to A99-ELP-R when coated at 4 ֯C. The
mean cell area of A99-ELP-R and A99-ELP-C, at 70 ֯C was 3062 and 3174 µm
2
with a 95% CI
[2937, 3186], [3032, 3315], respectively, compared to an average area of 2301 µm
2
with a 95% CI
[2179, 2423] when A99-ELP-R was coated at 4 ֯C. The major axis of A99-ELP-R and A99-ELP-
C at 0.5µM had a mean value of 73.89 and 76.45 µm with a 95% CI [72.16, 75.62], [74.88, 78.02],
23

respectively, compared to 63.16 µm with a 95% CI [61.72, 64.60] of A99-ELP-R coated at 4 ֯C.
Additionally, the conjugated version and recombinant construct were not statistically significantly
different from each other in cell area at 1 µM or major axis length at 0.5 µM and above. The cell
perimeter of A99-ELP-C and A99-ELP-R coated at 70 ֯C was not statistically significantly
different from A99-ELP-R coated at 4 ֯C until 1 µM (mean value of 264 µm with a 95% CI [254.4,
273.7], p = ≤ 0.001) where both the conjugated and recombinant versions began to have a larger
perimeter, with a mean value of 319.7 and 299.9 µm with a 95% CI [313.2, 326.1], [290.6, 309.1],
respectively.

Figure 8. Cell adhesion and morphology on various coated plates. Non-TC treated 96-well plates were
coated with 1 µM of peptides for 1 h at 4 or 70 ֯C except A99-chitosan (200 µM at RT overnight, according
to previously published studies) and after coating, 5x10
3
HDFs were plated and incubated at 37 ֯C for 2 h.
A) A99 coated peptides without any scaffold do not promote cell attachment and spreading and lead to non-
specific binding. B) I30 demonstrates some non-specific binding and spreading but very minimally. C)
A99-chitosan has already shown to have excellent cell attachment activity in previously published studies
and is used as a positive control. D) A99-ELP-R coated plates when incubated below the T t at 4 ֯C have
some cell attachment and spreading activity, greater than I30 by itself but less activity than observed in E
and F. E and F) Conjugated A99-ELP-C and recombinant A99-ELP-R when coated above the T t provides
an excellent scaffold and signal for cell attachment and spreading activity. Images converted to greyscale.
24

2.4.6. Visualizing cell spreading on A99-ELP-R coated plates
The cell spreading activity and integrin oligomerization was indirectly assessed by
immunofluorescent labeling of vinculin (Figure 9D). When 1 µM of A99-ELP-R is coated above
the Tt for 1 h, HDFs attach to the substrate as indicated by the strong staining of focal adhesions
with the anti-vinculin staining (green). In addition, distinct F-actin staining (red) using rhodamine
phalloidin demonstrates the relationship of the F-actin cytoskeleton to the focal adhesion sites.

25

Figure 9. Cell spreading activity. A99 only was coated at 70 ֯C (magenta) for 1 h, while recombinantly
purified A99-ELP-R was coated at 4 (black) or 70 ֯C (teal) for 1 h and A99-ELP-C (conjugated form) was
coated only at 4 ֯C (red) for 1 h in non-TC plates. 5x10
3
HDFs in 1% BSA containing DMEM were plated
and incubated at 37 ֯C for 2 h in 5% CO 2. A) The average cell area (µm
2
) over various concentrations of
peptide where A99-ELP-R and A99-ELP-C coated at 70 ֯C are statistically significantly larger compared to
A99-ELP-R coated at 4 ֯C at 0.5 µM and above (p = ≤ 0.0001). A99-ELP-C and A99-ELP-R coated at 70
֯C are not significantly different from each other. B) The average cell perimeter over different concentrations
demonstrating a statistically significant differences between the perimeter of A99-ELP-R, A99-ELP-C
coated at 70 ֯C and A99-ELP-R coated at 4 ֯C starting at 1 µM (p = ≤ 0.001). C) The major axis length or
largest distance between two opposite ends of the cells were measured (µm). At 0.5 µM, A99-ELP-R and
A99-ELP-C coated at 70 ֯C have a much greater distance between the longest ends compared to A99-ELP-
R coated at 4 ֯C (p = ≤ 0.0001). Error bars indicate the 95% confidence interval. D) Fluorescent micrographs
of HDFs grown on 1µM of A99-ELP-R coated plates in blocking media (0.1% BSA in DMEM) for 2 h at
37 ֯C. Focal adhesion points are indicated by the white arrowhead. Staining for F-actin using phalloidin
(594, red), anti-vinculin antibody (488, green) for focal adhesions and the nucleus (DAPI, blue). The green
channel was adjusted to have a higher intensity for ease of viewing.

26

2.5. Discussion
The emerging field of tissue regeneration and restoration has prompted the progression of
innovative technologies and biomaterials that restore, maintain, and improve damaged tissues.
(Dreher, Simnick et al. 2008, Janib, Gustafson et al. 2014, Dhandhukia, Shi et al. 2017) Thus,
improving synthetic biomaterials that facilitate the wound healing process by targeting the proper
stimuli (such as mechanical or recognition motifs) from the ECM have become widely explored.
This study introduces an approach that uses laminin α1 chain peptide, A99, as the
recognition motif and ELP as the scaffold in combination to promote cell adhesion and spreading
(Figure 10). The A99 (AGTFALRGDNPQG) RGD-containing sequence previously demonstrated
excellent cell adhesion activity and was the main candidate for this study. ELPs were selected as
the scaffold due to their biocompatibility, ease of purification, and ability to generate large
quantities of monodisperse, high-molecular weight polypeptides. We defined the Tt of A99-ELP-
R at 50 µM as 24 ֯C; however, our studies were conducted at the minimal concentration required
to promote cell attachment activity. At 0.2 µM of A99-ELP-R, the ELPs do not coacervate at 70
֯C and therefore do not efficiently coat to the plate. Alternatively, 0.5 µM of A99-ELP-R forms
coacervates at 70 ֯C and coats the wells promoting great cell attachment as shown in Figure 4A.
Additionally, our attempt to evaluate the A99-ELP-R coating efficiency led to equivocal results as
our various of methods to quantitate all A99-ELP-R was inconsistent when utilizing detergents
such as NP-40, Triton X-100, guanidine HCl or urea. The constructs were comprised of the fully
recombinant, A99-ELP-R fusion, which was directly compared to CGG-A99, a chemically
conjugated, A99-ELP-C version. A99-ELP-R produces a 100% homogenous product, which is
typically a valuable trait for upscaling and manufacturing; whereas A99-ELP-C chemical
conjugation produces a 50% yield, in which the unconjugated I30 would require additional purified
27

from A99-ELP-C by size exclusion or affinity chromatography. This study directly evaluated the
possibility that this chemical conjugate would have lower activity than the recombinant fusion.
Both constructs appeared to have similar activity cell attachment and spreading. Chemical
conjugation is extremely useful, owing to its ability to rapidly attach different moieties, serving a
different purpose. For example, a scenario would begin with an experimental procedure which
would first involve the simple 2-step process to conjugate chemically a cysteine-containing peptide
to ELP for rapid conjugation of various other peptide sequences. This allows the possibility to
screen a large library of synthetic peptides for bioactivity. Once the best candidate providing the
desired phenotype is found, that specific sequence could be inserted for recombinant DNA cloning
as an ELP-fusion for subsequent studies (Figure 7). The recombinant version could then be scaled-
up without the requirement for additional bioconjugation or purification of unconjugated peptides.
28

Figure 10. Schematic illustration of the strategy for engineering laminin bioactive peptide and ELPs
as a biomaterial to promote cell attachment and spreading. I30 is used as a scaffold and was fused to
laminin α1 chain, A99 (AGTFALRGDNPQG) bioactive RGD containing peptide sequence. This method
can be utilized for rapid screening for other biologically active peptides and produced in large yields. These
factors provide the key components for successful cell culture and tissue engineering.
29

Some differences between the fully recombinant construct and the chemically conjugated
version lie within its sequence (found on Table 1). The A99 sequence in the recombinant construct
follows N to C terminus similarly to that of the I30 sequence, yet the cysteine containing, CGG-
A99 peptide, after conjugation is in the C to N terminus orientation. Although this raises questions
whether this abolishes the activity of the peptide sequence, observation of the sequence shows that
the RGD remains in the appropriate N to C orientation for interaction with cell-surface receptors,
despite having an N to N-terminal conjugation with the I30. Another minor difference is the spacer
or linker in which the recombinant construct retains the original start codon methionine and the
conjugated version has a cysteine. Chemical conjugation of cysteine containing, CGG-A99 peptide
requires a chloroacetylation intermediate step of I30 to facilitate conjugation of the laminin peptide
and utilization of N-(chloroacetoxy)succinimide was chosen based on the authors’ expertise. Due
to the design of these two constructs, these minor differences cannot be ruled out as having an
effect of the differences in bioactivity between the two constructs.

Table 1. Summary of the amino acid sequences used.
Label Amino acid sequence Molecular Weight* (kDa) Total Yield
A99 AGTFALRGDNPQG 1.3 –
CGG-A99 cggAGTFALRGDNPQG** 1.5 –
I30 G(VPGIG) 30Y 12.94 20-35 mg/L
A99-ELP-C
(VPGIG) 30Y
cggAGTFALRGDNPQG**
14.5 50%
A99-ELP-R AGTFALRGDNPQGgmg(VPGIG) 30Y** 14.4 20-35 mg/L
* The expected molecular weight is based on the open reading frame for the expressed polypeptide and was
calculated using ExPASy computational pI/Mw tool.(Wilkins, Gasteiger et al. 1999)
** Lowercase letters indicates the amino acids in single-letter code comprising the spacer or linker.

30

A major characteristic of ELPs is that they are thermo-responsive and phase separate when
above the Tt. Since this process is reversible, ELPs can be rapidly purified from E. coli using
through cyclic induction of phase separation. ELPs can be modified at the DNA level to adjust the
temperature at which they form coacervates, which makes them tunable to the end-user’s
requirements. Traditional coating of bioactive peptides using biochemical approaches often require
overnight incubation periods. Recently previously published study using ELPs also coated the
materials overnight at 4 ֯C.(Jeon, Park et al. 2011) Utilizing the ELPs thermo-responsive
characteristics by heating up the plate above the Tt to induce coacervation and reduce coating time
seems to increase the effectiveness of cell attachment and spreading (Figure 11). The original
coating time for A99-ELP-R started overnight similarly to the previously published studies, but
while testing for coating efficiency, biological activity was shown to be possible as little as 5 min
of coating incubation time when above the Tt (Figure 8). In addition, coating A99-ELP-R below
the Tt for a significantly longer period of time could potentially achieve similar results to 1 h at 70
֯C. Then, while optimizing the experimental procedures for the appropriate concentration, the
preliminary data obtained showed that the total numbers of cells attached to the 96-well plate
plateaued after 30 min with around 0.5-1 µM of A99-ELP-R so 1 h was determined to be sufficient
coating time.

31

A re a (  m
2
)
A 9 9 -E L P -R
0 1 0 0 0 2 0 0 0 3 0 0 0
0 .2  M
0 .5  M
1  M
0 .2  M
0 .5  M
1  M
7 0  C
4  C
n .d .

Figure 11. Average area of HDFs at various coating concentrations of A99-ELP-R at 4 ֯C or 70 ֯C.
A99-ELP-R (0.2, 0.5, 1 µM) coated for 5 min in non-tissue culture treated 96-well plates with 5x10
3
HDFs
for 2h in triplicates performed three times. A99-ELP-R coated at 70 ֯C promotes greater cell spreading than
at 4 ֯C. There is a statistically significant difference in total area when comparing 70 ֯C vs 4 ֯C of the same
concentration of A99-ELP-R coated plates (p = ≤ 0.0001). Error bars indicate 95% confidence interval. n.d.
= not detectable.

Cell attachment on A99-ELP-R coated plates shows that the minimum required
concentration of A99-ELP-R to have adhesion activity is between 0.2 and 0.5 µM as demonstrated
in Figure 4A. Although some cell adhesion occurs at 0.2 µM, there is no substantial spreading.
With only a slight increase in the incubation concentration to 0.5 µM, substantially more cell
attachment and spreading activity was observed. This was also the case with A99-ELP-C coated
plates when determining the area of the cells. 0.2 µM of coated A99-ELP-R and less had some cell
attachment, but the cells were all spherical in shape, so they were determined to be non-specifically
adherent. Conversely, HDFs cultured on A99-ELP-R coated plates at concentrations greater than
10 µM (up to 1000 µM), show that the crystal violet may stain the A99-ELP-R coat, making it
more difficult to visualize and measure the cells. In conclusion, the data presented suggests that
A99-ELP-R and A99-ELP-C coated at 70 ֯C can be used interchangeably for their impact on cells
32

attachment, and their impact on characteristics of attached cells such as the area of cells, maximum
axis length of cells and the perimeter of the cells. This method also provides a rapid way to screen
for biologically active peptides, and the coating time can be short compared to traditional
biochemical approaches for coupling and coating.

2.6. Summary
In this chapter, a proof of principle involving hydrophobic ELP, I30, and a laminin peptide, A99,
was evaluated for its usefulness in cell culture engineering. Both the chemically conjugated and
recombinant versions of A99-ELP had similar bioactivity and excelled when coated above the Tt.

2.7. Acknowledgements
AT would like to thank Drs. Anna Kiyomi and Nobuhito Hamano for their moral support in this
research. This work was made possible by the Japan Society for the Promotion of Science (JSPS)
(AT, PE18045) and the JSPS KAKENHI (YK, 17K07180. MN, 18K06637) grant. JA thanks the
Gavin S. Herbert Endowed Chair of Pharmaceutical Sciences.
33

CHAPTER 3: LAMININ-DERVIVED ELPs OPTIMIZE CELL
SPREADING

3.1. Abstract
A major component of the extracellular matrix (ECM), laminins, modulate cells via diverse
receptors. Their fragments have emerging utility as components of ‘ECM-mimetics’ optimized to
promote cell-based therapies. Recently we reported that a bioactive laminin peptide known as A99
enhanced the cell-binding and spreading via fusion to an elastin-like polypeptide (ELP). The ELP
‘handle’ serves as a rapid, non-covalent strategy to concentrate bioactive peptide mixtures onto a
surface. We now report that this strategy can be further generalized across an expanded panel of
additional laminin-derived elastin-like polypeptides (LELPs). A99 (AGTFALRGDNPQG),
A2G80 (VQLRNGFPYFSY), AG73 (RKRLQVQLSIRT) and EF1m (LQLQEGRLHFMFD)
promote cell spreading and show morphologically distinct F-actin formation. Equimolar mixtures
of A99:A2G80-LELPs have synergistic effects on adhesion and spreading. Finally, three of these
ECM-mimetics promote neurite outgrowth of PC-12 cells. The evidence presented here
demonstrates the potential of ELPs to deposit ECM-mimetics with applications in regenerative
medicine, cell therapy, and tissue engineering.

3.2 Introduction
Tissue regeneration is a complex biological phenomenon orchestrated by many components from
the extracellular matrix (ECM). The ECM is comprised of an extensive macromolecular network
that provides structural scaffolds and multi-functional signaling molecules that promote growth
34

and differentiation (Badylak 2002, Domogatskaya, Rodin et al. 2012, Yue 2014). Tissue injuries
resulting from trauma are generally repaired through the natural wound healing process; however,
severe damage requires medical intervention. To restore the function of damaged tissues, the field
of tissue engineering has focused effort on synthetic biomaterials that emulate the native ECM
(Heilshorn, DiZio et al. 2003, Mie, Mizushima et al. 2008, Cai and Heilshorn 2014, Fernandez-
Colino, Arias et al. 2014, Kakinoki and Yamaoka 2014, Hwang, Sullivan et al. 2020). ‘ECM-
mimetics’ comprised of laminin peptides address some drawbacks of other technologies and are a
promising strategy for regenerative medicine applications (Yamada, Hozumi et al. 2010, Jeon
2013, Kim, Jekarl et al. 2014, Fujimori, Kumai et al. 2017).
The ECM consists of collagen, fibronectin, elastin, laminin and a variety of other
proteoglycans. While collagen is the most abundant structural matrix protein, laminins contribute
significantly to tissue development, regeneration and homeostasis (Hamill, Kligys et al. 2009,
Domogatskaya, Rodin et al. 2012, Riederer, Bonomo et al. 2015). Laminins are large
heterotrimeric, cross-shaped glycoproteins comprised of an α, β and γ chain. They interact with a
variety of cell surface receptors, such as integrins, syndecans and dystroglycans. While laminins
do provide structural support in the ECM, they also play important roles in cell signaling for
adhesion and migration. Thus far, 19 isoforms have been identified from: five α-chains, three β-
chains, and three γ-chains (Durbeej 2010). These isoforms are variably expressed throughout the
body at different developmental stages; however, the biological relevance of many of these
isoforms are not yet well-defined. Several groups have identified laminin expression dysregulation
in a variety of epithelial disorders, including congenital muscular dystrophy (α2), junctional
epidermolysis bullosa (α3, β3 and γ2) and Pierson syndrome (β2), (Aberdam, Galliano et al. 1994,
Uitto, Pulkkinen et al. 1997, Allamand and Guicheney 2002, Chen, Zhou et al. 2013, Theocharis,
35

Manou et al. 2019). Therefore, since they contribute to diverse biological roles such as cell
adhesion and migration, differentiation and angiogenesis, laminin-derived peptides may have
applications in cell therapy and tissue repair.
Several laminin chains from Mus musculus were previously screened for biological activity
through a systematic process involving a synthetic peptide library that spanned the entire amino
acid sequence. Peptides that were approximately 12 amino acids in length with an overlap of four
amino acids were screened for cell adhesion activity and morphology. A complete table of
bioactive laminin peptides were tabulated (Nomizu, Kim et al. 1995, Nomizu, Kuratomi et al.
1997, Nomizu, Kuratomi et al. 1998, Makino, Okazaki et al. 2002, Suzuki, Nakatsuka et al. 2003,
Suzuki, Hozumi et al. 2010). Many of these peptides had considerable biological activity; however,
the candidates for this study were selected based on their induction of distinct and easily evaluated
morphologies. Specifically, the RGD-containing A99 (AGTFALRGDNPQG) from the laminin α1
chain binds to integrin αvβ3, which has often been shown to promote cell attachment. A2G80
(VQLRNGFPYFSY) from the laminin α2 chain binds to specifically to α-dystroglycans. AG73
(RKRLQVQLSIRT) from laminin α1 binds to syndecans, promoting cell adhesion with a
membrane ruffling morphology. Also from laminin α1, EF1m (LQLQEGRLHFMFD) promotes
some cell attachment and is a truncated form of longer peptide EF1 developed to enhance
solubility. EF1m binds to integrin α2β1 and induces an elongated morphology.
The characteristics of an ideal biomaterial for rapid wound healing and tissue regeneration
would have biological and cellular responses that closely mimic the natural ECM. Many strategies
have employed synthetic or naturally occurring polymers such as poly(N-isopropylacrylamide),
poly(lactic acid), poly(glycolic acid), sodium alginate, gelatin, hyaluronan or chitosan as scaffolds
and bioconjugate chemistry to attach small, biologically active peptides (Kim and Park 2006,
36

Yamada, Hozumi et al. 2013, Kakinoki, Nakayama et al. 2014, Fujimori, Kumai et al. 2017, Su,
Satchell et al. 2019). While these approaches have been successfully optimized over some time for
conjugation under mild conditions, the homogeneity and labeling efficiency have been a focus on
an area for improvement. Alternatively, recombinant expression in E. coli can produce
biomaterials with high precision and relatively low cost. We have adopted this strategy to design
a small library of fusions using laminin peptides linked to an elastin-like polypeptide (ELP), which
was bio-inspired from another ECM protein called tropoelastin.
ELPs are thermo-responsive biopolymers with favorable biophysiochemical properties as
a scaffold for tissue engineering (Nettles, Chilkoti et al. 2010, Nettles, Haider et al. 2010). ELPs
are attractive because they are biosynthesized with high fidelity with respect to their amino acid
sequence, molecular weight and polydispersity (Chilkoti, Christensen et al. 2006, Roberts,
Dzuricky et al. 2015, Despanie, Dhandhukia et al. 2016, Varanko, Su et al. 2020). ELPs are
typically composed of pentapeptide repeats consisting of (Val-Pro-Gly-Xaa-Gly)n, where Xaa is any
guest residue amino acid and n is the number of repeats. Interestingly, previous research had
demonstrated that the hydrophobic amino acid repeat domains interact with elastin binding
proteins and integrin αv 5 contrary to not having the RGD motif (Lee, Bax et al. 2014, Lee, Yeo
et al. 2017, Yeo and Weiss 2019). Moreover, to the best of our knowledge, the first successful ELP
fusions designed as ECM-mimetics were linked to the CS5 domain peptide from fibronectin
(Heilshorn, DiZio et al. 2003). These biopolymers undergo a reversible inverse phase separation
from aqueous solution above the lower critical solution temperature (LCST), also known as the
transition temperature (Tt). As for synthetic LCST polymers, ELP ‘coacervation’ is promoted by
the organization of water molecules around hydrophobic side-groups, such as those on valine or
isoleucine. Upon heating, the entropic cost of this ordered water rises until the free energy of
37

mixing becomes unfavorable. Above the Tt, ELP coacervation is accompanied with a transition of
the peptide backbone from random coils to form more-ordered secondary structures (type II β-
turns, β-spirals or distorted β-sheets) (Li, Garcia Quiroz et al. 2014, Pastuszka and MacKay 2016).
This reversible ELP-mediated phase separation serves a dual role in triggered assembly as well as
protein purification. These unique characteristics have led to innovative ELP-biomaterials for
tissue and cell engineering (Raphel, Parisi-Amon et al. 2012, Benitez, Mascharak et al. 2016,
Mozhdehi, Luginbuhl et al. 2018).
We have recently characterized a single laminin peptide fused to ELPs both chemically
and recombinantly and demonstrated its usefulness as a scaffold (Truong, Hamada et al. 2020).
This work expands the library of ELP laminin peptides to include A2G80 (VQLRNGFPYFSY),
AG73 (RKRLQVQLSIRT) and EF1m (LQLQEGRLHFMFD) each linked to an ELP known as
I30 (VPGIG)30. In comparison with A99 (AGTFALRGDNPQG), this chapter evaluates their
ability to promote cell attachment, spreading and differences in cell morphology. Additionally, we
show that minimal concentrations of specific peptides mixed in a 1:1 ratio have a synergistic effect
and that some of the LELPs provide biological cues that induce neurite outgrowth of PC-12 cells.
This study overall demonstrates the usefulness of these laminin peptides in conjugation with ELPs
as a biomaterial for tissue engineering.

3.3. Materials and methods
3.3.1. Construction, expression and purification of ELPs
I30 (VPGIG)30 was generated in a pet25b (+) vector using traditional cloning that is compatible
with recursive directional ligation by plasmid reconstruction (Pre-RDL) as previously described
38

(McDaniel, Mackay et al. 2010, Janib, Pastuszka et al. 2014). The nucleotide sequences
corresponding to mouse laminin chains for A99 (AGTFALRGDNPQG), A2G80
(VQLRNGFPYFSY), AG73 (RKRLQVQLSIRT) and EF1m (LQLQEGRLHFMFD) were
obtained by a BLAST query search (https://blast.ncbi.nlm.nih.gov/Blast.cgi). All synthetic
oligonucleotides (Eurofins Genomics, Tokyo, Japan) were constructed in the same manner to
allow placement of the laminin peptides on the N terminus of I30 similarly as previously described
(complete oligonucleotide sequences and explicit details may be found in Table 2) (Guo, Lee et
al. 2018).

Table 2. Cassette of oligonucleotide sequences of the bioactive laminin peptides flanked with
NdeI, BseRI and BamHI restriction recognition sites for recursive directional ligation by
plasmid reconstruction into pet25b (+) vector.
Label Nucleotide sequence*
A99 atacatatggccggcacctttgccttgcgaggggacaaccctcaaggcggtggttactgatctcctcggatccatc
A2G80 atacatatggttcagctgaggaatggcttcccctacttcagttatggtggttactgatctcctcggatccatc
AG73 atacatatgcggaagaggctccaggtgcagctgagcattcggacaggtggttactgatctcctcggatccatc
EF1m atacatatgctccagctccaagagggccgcctgcacttcatgtttgatggtggttactgatctcctcggatccatc
* Bolded is the final nucleotide sequence inserted into vector I30

In addition, two glycine amino acids were placed between the laminin peptide and I30 as
a minimal spacer. Restriction sites containing NdeI (N-termini) and BseRI/BamHI (C-termini)
were flanked between the laminin peptide nucleotide sequences. The oligonucleotides were
inserted into an empty pet25b (+) vector acting as a donor by digesting restriction sites NdeI and
BamHI (R0111S, R3136S, all enzymes were from New England Biolabs, Ipswich, MA). The
39

donor plasmids containing the laminin peptide sequence insertion were digested with EcoRV and
BseRI (R3195S, R0581S) while the recipient plasmid containing the I30 sequence were cut with
NdeI and EcoRV. The fragments containing either the laminin peptide or I30 sequence were then
ligated with T4 DNA ligase (M0202S) overnight at 16 °C. Successful cloning was verified by
Sanger sequencing (Genewiz, Tokyo, Japan). All fusions are named with their respective laminin
peptide label with ELP designated at the end (e.g., laminin A99 peptide fused to I30 is A99-ELP.
Refer to Table 3 for complete nomenclature).

Table 3. Recombinant laminin-derived elastin-like polypeptides evaluated in Chapter 3
Label Amino acid sequence
1
Expected MW
2
(kDa)
Measured MW
3

(kDa)
T t
4
(°C)
I30 G(VPGIG) 30Y 12.94 12.94 25.6
A99-ELP AGTFALRGDNPQGGG(VPGIG) 30Y 14.29 14.28 27.7
A2G80-ELP VQLRNGFPYFSYGG(VPGIG) 30Y 14.47 14.49 15.7
AG73-ELP RKRLQVQLSIRTGG(VPGIG) 30Y 14.48 14.62 20.5
EF1m-ELP LQLQEGRLHFMFDGG(VPGIG) 30Y 14.62 14.74 12.5
1
Underline letters indicate amino acids in single letter code comprising the spacer.
2
The expected molecular weight is based on the open reading frame for the expressed polypeptide and was calculated using ExPASy
computational pI/Mw tool.
3
The molecular weight was independently confirmed for the major species using matrix-assisted laser desorption ionization mass spectrometry.
4
The transition temperature, T t, is based on 10 µM of fusion polypeptide.

Transformed E. coli was grown in terrific broth (1 L contained 12 g tryptone, 24g yeast
extract, 4 ml glycerol, 2.31 g of KH2PO4 and 12.54 g K2HPO4) and incubated in a shaker at 250
RPM and 37 °C overnight for approximately 16 – 18 h. The medium was spun down at 4000 g for
10 min, supernatant discarded and thoroughly resuspended in 30 ml ice-cold phosphate-buffered
saline (PBS) in a 50 ml conical tube. The cells were lysed by sonication (S-4000, Misonix,
40

Farmingdale, NY) on ice and prechilled water for 10 minutes, then excess debris were removed
by adding 250 µl of poly-ethyleneimine (PEI, 408700-1L, Sigma-Aldrich, St. Louis, MO) and
affixed on a spinning rotor (346 hematology/chemistry mixer, Fisher Scientific, Waltham, MA)
for 10 min at 4 °C. The debris was removed by centrifugation for 15 min at 4 °C, 4000 g (cold
spin) and the supernatant containing the fusions were induced to phase separate by the addition of
1 M sodium chloride and heating the sample in a water bath at 37 °C for 10 min. This was followed
by centrifugation at 37 °C for 15 min, 4000 g (hot spin), whereby the supernatant was discarded,
and the thin pellet carefully resuspended and solubilized in 6 ml of ice-cold PBS. The cold and hot
spins were performed 3 times to ensure purification of particulates from E. coli. However, during
the last hot spin, the pellet was resuspended in 2 ml of ice-cold PBS to increase the overall
concentration, aliquoted to 2 ml microcentrifuge tube and stored at -20 °C until ready for use. The
expected amino acid sequences and molecular weights (MW) are: I30 (G(VPGIG)30Y, 12.94 kDa),
A99-ELP (AGTFALRGDNPQGG(VPGIG)30Y, 14.29 kDa), A2G80-ELP
(VQLRNGFPYFSYGG(VPGIG)30Y, 14.47 kDa), AG73-ELP
(RKRLQVQLSIRTGG(VPGIG)30Y, 14.48 kDa) and EF1m-ELP
(LQLQEGRLHFMFDGG(VPGIG)30Y, 14.62 kDa).
The plasmid DNAs containing the fusion constructs were amplified with DH5α or One
Shot TOP10 chemically competent cells (18265017, C404003, ThermoFisher Scientific, Waltham,
MA) and then transformed in BLR (DE3) competent cells (69053-3, Millipore Sigma, Burlington,
MA) for protein expression. LELPs were purified by ELP phase separation as described previously
(Meyer and Chilkoti 1999, Truong, Hamada et al. 2020). The purity and molecular weights were
determined by SDS-PAGE using a 4% stacking gel and 15% resolving gel then stained briefly
with Coomassie Brilliant Blue R (BP101-25, Fisher Scientific, Waltham, MA). Refer to Table 4
41

for the yield and purity of the LELPs. The LELPs MWs were verified by MALDI-TOF (matrix-
assisted laser desorption/ionization- time of flight, rapifleX, Bruker, Billerica, MA).

Table 4. Estimated purification yield and purity based on SDS-PAGE.
Polypeptide Total yield
1
(mg/L) Purity
2

I30 20 – 33 84.5%
A99-ELP 20 – 38 87.4%
A2G80-ELP 20 – 40 96%
AG73-ELP 20 – 32 92.2%
EF1m-ELP 20 – 23 80.4%
1
Range of yields in terms of purified mg of ELP per liter of
bacterial culture media.
2
Purity is measured by dividing the integrated density of the
protein band by the integrated density of the total bands in each
column.

The laminin peptide-ELP concentrations were determined by diluting them 1:1 or 1:2 in 6
M guanidine HCl for the unfolding of the polypeptide and the optical density (OD) measured at
280 nm with a 1 mm light path using a spectrophotometer (NanoDrop 2000, ThermoFisher
Scientific, Waltham, MA). A 350 nm value of less than 0.015 for each 280 nm reading guaranteed
unfolding and was accepted. This measurement was done in triplicates and averaged. The Beer-
Lambert law was applied to calculate the concentration:
𝐸𝐿𝑃 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑀 ) =
𝑂𝐷
280
× 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 ε × l (cm)

The estimated molar extinction coefficient, ε, for A99-ELP, AG73-ELP and EF1m-ELP are 1285
(M
-1
cm
-1
) and A2G80-ELP is 3855 (M
-1
cm
-1
). These values were calculated based on the number
of tyrosine amino acid residues in 6 M guanidine HCl.
42

3.3.2. Measuring the hydrodynamic radius of the LELPs.
The hydrodynamic radius (Rh) of the LELPs were measured at 7 C and 32 C. Briefly, 60 µl of
10 µM LELP was loaded in triplicates into a 384 black well, clear bottom plate (781906, Greiner
Bio-One, Monroe, NC) and 15 µl of mineral oil added to the top. The plate was spun down at
1000g for 1 min and was analyzed with the DynaPro plate reader (Wyatt Technologies, Santa
Barbara, CA).
3.3.3. Defining the lower critical solution temperature
The thermal phase behavior of A99-ELP, A2G80-ELP, AG73-ELP and EF1m-ELP was
determined by monitoring the OD at 350 nm with a temperature-controlled spectrophotometer
(DU/800 UV-vis, Beckman Coulter, CA). Tm microcuvettes (523878, Beckman Coulter, CA)
were prechilled to 10 – 11 °C in the spectrophotometer and 250 µl of 10 µM of sample was
carefully pipetted into the microcuvettes. These samples were left to equilibrate for 20 min before
beginning the absorbance measurements. The measurements were taken as a function of
temperature at 1 °C/min where the LCST is defined when the maximum first derivative of the OD
at 350 nm is achieved. Additionally, the Tt was also determined as a function of concentration (50,
25, 12.5, 6.25, and 3.125 µM).
3.3.4. Cell culture
Neonatal human dermal fibroblasts (HDFs) (106-05n, Cell Applications Inc., San Diego, CA) were
maintained in basic DMEM (11885084, ThermoFisher Scientific, Waltham, MA) supplemented
with 10% FBS (35-011-CV, Corning, Corning, NY). Rat pheochromocytoma cells (PC-12) (CRL-
1721.1, American Type Culture Collection (ATCC), Manassas, VA) were maintained in RPMI
(30-2001, ATCC, Manassas, VA) supplemented with 5% FBS and 10% horse serum (26050088,
43

ThermoFisher Scientific, Waltham, MA). Both cell lines were maintained at 37 °C in 5% CO2
humidified tissue culture incubator.
3.3.5. Determining the cell adhesion and spreading activity
The attachment and spreading activities were assessed at various coating concentrations (0.01, 0.1,
0.5, 1, 10 µM) of the LELP fusions. These were coated in 96-well non-tissue culture treated,
polystyrene plates (701011, Nest Biotechnology, Wuxi, China) in triplicates for 1 at 37 °C with a
digital dry heating block (IC25, Torrey Pines Scientific, Carlsbad, CA). After incubation, the wells
were washed 3 times with room temperature (RT) PBS and 2x10
3
(cell spreading) or 2x10
4
(cell
attachment) HDFs were plated into each well in 0.1% bovine serum albumin (BSA) in basic
DMEM (no serum). The cells were incubated at 37 °C in the tissue culture incubator for 1 h (cell
attachment) or 2 h (cell spreading) and after, fixed and stained with 0.5% crystal violet in 10%
methanol for 2 min. Wells were subsequently washed with ample distilled water and allowed to
dry facedown overnight. Brightfield microscopy images were collected (Cytation 5, BioTeK,
Winooski, VT) using flat field optical, fluorite aberration corrected, phase contrast 10x, 20x and
40x Olympus objective lens (numerical aperture of 0.3, 0.45 and 0.6 respectively). Briefly, four
random areas (using the same fixed offset for every well) were automatically obtained via Gen5
software (3.0.5.11, BioTeK, Winooski, VT) within each well, within all the triplicates, and the
experiments were performed a minimum of 3 times on different days. After collection of all the
images, the number of cells attached were manually scored or the area, perimeter and maximum
length was analyzed using CellProfiler. An improved pipeline with greater accuracy was modified
from our previous pipeline, and the general steps are explained in sequential order: metadata
containing the date, experiment number, laminin peptide-ELP construct, and concentration were
extracted and the background illumination was corrected. To reduce the background noise,
44

suppressing features was applied to reduce speckles. The region of interests (ROIs) were
automatically identified using the Otsu thresholding algorithm and each image was manually
verified for correct ROI identification. Any objects touching the borders of the images were
discarded and any incorrect object selected was manually adjusted. The pipeline is publicly
available for use. (https://github.com/intekmda/LamininELP_CellSpreading)
3.3.6. Evaluating various concentration ratios and their cell adhesion activity
Concentrations of 0.25 and 0.5 µM each in a 1:1 mixture of every combination of A99, A2G80,
AG73 and EF1m were evaluated for cell adhesion activity. Plates were coated as previously
described above for 1 h at 37 °C. 5x10
3
HDFs were seeded and incubated at 37 °C for 2 h.
Additionally, A99:A2G80-ELPs were coated and 2x10
4
HDFs were seeded and incubated at 37 °C
for 1 h for direct comparison with the other cell attachment experiments. The cells were fixed with
crystal violet as previously mentioned and 4 images were taken per well in triplicates completed
at least 3 times on 3 different days. The number of cells were manually scored for tabulation. Cells
at the border of the images were included in the count.
3.3.7. Inhibition assay
To inhibit HDFs from attaching and spreading to the LELP coated 96-well plates, HDFs were
trypsinized and resuspended in 0.1% BSA containing basic DMEM with no serum and pretreated
with 5 mM EDTA (ethylenediaminetetraacetic acid) (S-311-500, Fisher Scientific, Waltham, MA)
or 10 µg/ml heparin sulfate (H4784-1g, Sigma-Aldrich, St. Louis, MO) for 15 min at 37 °C. 2x10
4

HDFs were seeded in 0.5 µM LELP coated wells and incubated at 37 C in 5% CO2 humidified
tissue culture incubator for 1 h. Cells were then stained with 0.5% crystal violet for 2 min, washed
with plenty of deionized water and allowed to dry before the qualitative analysis.
45

3.3.8. Immunofluorescence staining and confocal microscopy
1 µM of the LELPs were coated onto black 96-well non-treated, flat and clear bottom, polystyrene
plates (655096, Greiner Bio-One, Kremsmünster, Austria) similarly as described above for 1 h at
37 °C. The plates were similarly prepared with RT PBS washes and 5x10
3
HDFs were seeded with
0.1% BSA in DMEM (no serum) in each well in triplicates. They were incubated for 2 h at 37 °C
in 5% of CO2 humidified cell culture incubators. Afterwards, the cells were fixed directly onto the
plates with 4% paraformaldehyde for 10 min, quenched with 50 mM ammonium chloride for 5
min, and permeabilized with 0.1% triton X-100 for 15 min. Each step included a 5 min RT PBS
washing step trice. The wells were blocked with 1% BSA in PBS for 30 min and incubated with
1:200 of mouse monoclonal anti-vinculin antibody (V9264-25UL, Sigma-Aldrich, St. Louis, MO)
overnight at 4 °C. The following day, the wells were incubated with 1:100 of secondary anti-mouse
Alexa Fluor 647 antibody (A21463, ThermoFisher Scientific, Waltham, MA) in 1% BSA for 1 h
at RT. Subsequently, rhodamine phalloidin (R415, ThermoFisher Scientific, Waltham, MA)
dissolved in methanol was incubated as 5 µl per 200 µl 1% BSA in PBS for 20 min at RT and was
finally incubated with DAPI (1mg/ml) for 5 min. An ample amount of RT PBS washing was
performed between all steps. The wells were voided of any remaining liquid and a thin layer of
ProLong antifade (P36961, ThermoFisher Scientific, Waltham, MA) was applied (approximately
1-2 drops). The 96-well plate was stored in aluminum foil at 4 °C until ready for imaging. The
stained HDFs were imaged using a confocal laser scanning microscope (LSM880, Carl Zeiss AG,
Jena, Germany) with super-resolution Airyscan (63x objective lens with a 1.4 aperture, plan-
apochromatic, infinity color corrected lens) in Immersol 518 F (444970-9000-000, Carl Zeiss
Microscopy, White Plains, NY).

46

3.3.9. Cell adhesion and neurite outgrowth
PC-12 cell neurite outgrowth was assessed in 96-well plates coated with 0.5 µM of A99, A2G80,
AG73, EF1m-ELPs and I30. Plates were prepared as previously described. At day 0, PC12 cells
were briefly resuspended and primed in fresh RPMI media containing 100 ng/ml of nerve growth
factor beta ( -NGF) and incubated at 37 °C in 5% of CO2 humidified cell culture incubator.
Approximately 24 h later, the cells were resuspended in advanced DMEM/F-12 media (12634010,
ThermoFisher Scientific, Waltham, MA) containing 100 ng/ml of -NGF, 20 nM of progesterone,
and was plated at 5x10
3
cells/well. They were incubated at 37 °C for an additional 96 h. Fresh
advanced DMEM/F-12 media with 100 ng/ml -NGF and 20 nM of progesterone was changed 48
h post seeding. Images were taken with the Cytation 5 instrument. PC-12 cells were scored for
adhesion and neurite outgrowth. Neurite outgrowth was considered to be positive if the neurites
were at least 3 times the length of the body.
3.3.10. Statistics
All experiments requiring statistics here were replicated a minimum of three times in triplicates.
The data is presented as the mean ± SD or CI. The statistical analyses were performed with
GraphPad Prism 8 (http://www.graphpad.com, La Jolla, CA). For all the analyses, the groups were
compared by the Kruskal-Wallis test. A Mann-Whitney t-test was used to compare the LELP ratios
between 0.25 versus 0.5 µM. A P value of 0.05 or less was considered to be statistically
significantly different.

47

3.4. Results
3.4.1. Generation of a library of thermo-responsive biologically active LELPs
The fusion of A99, A2G80, AG73 and EF1m peptide sequences to I30 was accomplished by
standard endonuclease restriction digestion, ligation and selection of transformed bacterial
colonies. The robust and simple cloning method with PRe-RDL compatible ends uses two halves
of plasmids for direct sequence insertion without the addition of extraneous nucleotides into either
the laminin peptide or the ELP, which retains the amino-terminal restriction site (Figure 12A).
Figure 12B) contains a flow diagram of the process whereby oligonucleotide ‘cassettes’
containing the laminin peptide sequences are inserted into a ‘donor’ pet25b(+) vector and the
halves are ligated. The plasmid nucleotide sequences and appropriate reading frame were verified
by Sanger sequencing. LELP plasmids and plasmid maps will be deposited and made available
from Addgene (http://www.addgene.org/Andrew_MacKay).

48

Figure 12. Construction of plasmid to express LELPs. A) Nucleotide and amino acid sequence of A99
(AGTFALRGDNPQG) peptide fused to I30 (all other constructs follow this design). The ELP gene encodes
a pentameric repeat of (Val-Pro-Gly-Ile-Gly) 30; however, only a partial sequence is shown. B) The flow
diagram demonstrates how the cloning is scheme compatible with directional recursive ligation by plasmid
reconstruction (PRe-RDL) to generate the complete library of laminin peptide-ELP plasmids. Laminin
peptide sequences flanking NdeI, BseRI and BamHI restriction sites are inserted into a ‘donor’ pet25b(+)
vector, then the segment (in red) is ligated with the amino terminus of an existing I30 plasmid. This
illustrates the cloning strategy, which yields a single, directional insertion of laminin peptide at the amino
terminus, which leaves no extraneous nucleotides between laminin peptide and I30.

49

3.4.2. Protein purification and characterization
The LELP library was prepared by recombinant protein production in E. coli and purified by ELP-
mediated phase separation. The MW and purity of I30 alone or fused with A99, A2G80, AG73 or
EF1m were verified by SDS-PAGE with a Coomassie stain (Figure 13A) and MALDI-TOF
(Figure 14). The measured MW’s are 12.94, 14.28, 14.63, 14.62 or 14.74 kDa respectively, which
match the expected MWs based on their amino acid sequence. The hydrodynamic radius (Rh) of
the LELPs are in Figure 15. The amino acid sequence of the laminin peptides fused to I30 have a
profound effect on the temperature at which the LELPs form coacervates. The Tt was measured
using 10 µM of LELP based on the occurrence of the maximum first derivative. A99-ELP, A2G80-
ELP, AG73-ELP and EF1m-ELP has a transition temperature of 27.7, 15.7, 20.5 and 12.5 °C,
respectively (Figure 13B). Additionally, a serial dilution was plotted as a function of concentration
demonstrating increases in Tt for I30, A99-ELP, A2G80-ELP and AG73-ELP in a log-linear
manner; however, the Tt for EF1m-ELP remained concentration independent (Figure 13C).
Complete details of the phase diagrams can be found in Figure 16.

50

Figure 13. Identity and phase behavior for LELP fusions. A) SDS-PAGE of purified I30 (12.9 kDa),
A99 (14.3 kDa), A2G80 (14.5 kDa), AG73 (14.5 kDa) and EF1m (14.6 kDa) ELP fusions after 3 rounds of
ELP-mediated separation/purification. Stained with Coomassie Brilliant Blue. MW confirmed with
MALDI-TOF (Table 3). B) The optical density measured as a function of temperature (1 °C/min) for 10
µM of each LELP and PBS only (control). C) The transition temperature as a function of concentration for
each LELP and I30.
51

Figure 14. MALDI-TOF results. Approximately 1mg/ml of the LELPs were analyzed by MALDI-TOF.
The data suggests that there may be some adducts that might be coming from the buffer in the solution or
the MALDI-TOF matrix.

52

0.01 0.1 1 10 100 1000
0
20
40
60
80
100
Rh (nm)
Intensity %
A99-ELP (32 C)
A2G80-ELP (32 C)
AG73-ELP (32 C)
EF1m-ELP (32 C)
I30-ELP (7 C)
EF1m-ELP (7 C)
AG73-ELP (7 C)
A2G80-ELP (7 C)
A99-ELP (7 C)

Figure 15. Hydrodynamic radius (Rh) of the LELPs. Dynamic light scattering analysis show 10 µM of
LELPs below the T t at 7 C, that the Rh is between 36 to 45 nm while above the Tt at 32 C, the Rh is
between 253 to 460 nm.

53

Figure 16. Measured transition temperature of the laminin peptide-ELPs. The optical density of (A)
I30 only, (B) A99, (C) A2G80, (D) AG73 and (E) EF1m-ELPs at 50, 25, 12.5, 6.25 and 3.125 µM were
monitor as a function of concentration over increasing temperature. Black dotted line is PBS only as a
control. EF1m-ELP’s OD begins to decrease at 25.7 C and may be explained by Figure 25.

54

3.4.3. Cell adhesion activity of LELP coated plates
Cell attachment activity was determined at various coating concentrations (Figure 17). A99-ELP,
A2G80-ELP, AG73-ELP and EF1m-ELP all demonstrated significant cell adhesion activity above
0.5 µM compared to I30 only (P < 0.0001). Below 0.5 µM, there was little cell attachment;
furthermore, attachment below this level resulted in round cellular morphology. At 1 and 10 µM,
coated A99-ELP and AG73-ELP both have maximal cell adhesion activity that is significantly
greater than the other LELPs and I30 only (P < 0.005). Non-laminin peptide cell attachment was
also observed above 1 µM with the I30 control, which is similar to that for A2G80-ELP and EF1m-
ELP. Interestingly at 10 µM, dishes coated with A2G80-ELP lose bioactivity, significantly
preventing cell adhesion compared even to I30 only (P < 0.0001).

Figure 17. Dose-dependent cell adhesion of LELP coated plates. A99, A2G80, AG73 and EF1m -fused
ELPs or I30 alone were coated in 96-well non-tissue culture treated plates for 1h at 37 °C, then 2x10
4
HDFs
were seeded and incubated in blocking media at 37 °C for 1 hour. Cell adhesion significantly increases at
0.5 µM for all coated LELPs (P < 0.005) compared to I30 only. A99-ELP and AG73-ELP coated plates
show significantly more cell attachment at 1 and 10 µM compared to I30 only. Coated A2G80-ELP
however, begins to lose attachment at 1 µM and is nearly abolished at 10 µM. Coated EF1m-ELP performs
similar to I30 only at 1 and 10 µM. Error bars represents SD.
55

3.4.4. Cell spreading and morphology on LELP coated plates
As an alternative strategy to compare cellular bioactivity, primary cell line HDF morphology was
also assessed at the same concentrations for cell adhesion (Figure 18). Similarly to the cell
adhesion, at concentrations below 0.5 µM, the average cell area, perimeter and maximum axis
length were minimal, which may be interpreted as non-specific cell attachment. However,
immediately at a coating concentration if 0.5 µM, there was a significant increase in these
morphological parameters compared to 0.1 µM. This was consistent with successful cell adhesion
and the promotion of cell spreading. A99, A2G80 AG73 and EF1m-ELPs area mean significantly
increased (P < 0.005). Additionally, A99 and AG73-ELPs significantly increased the area mean
from 0.5 to 1 and to 10 µM (P < 0.0001). EF1m-ELP plateaued in activity above 0.5 µM of coating
(P > 0.05). Surprisingly, A2G80-ELP at 10 µM lost most of its bioactivity (Figure 3A). The
perimeter of A99, A2G80, AG73 and EF1m-ELPs also demonstrated significant differences from
0.1 to 0.5 µM (P < 0.05). The same trend followed for A99 and AG73-ELPs where the perimeter
significantly increased also at 1 and 10 µM (P < 0.005). There was a significant difference in
perimeter for EF1m-ELP between 0.5 and 1 µM (P < 0.005), however, there was no significant
difference from 1 to 10 µM. A2G80-ELP at 10 µM also has a significant drop in the cell’s
perimeter, consistent with its decrease in its area (Figure 3B). The maximum axis length which
measures the furthest point across each cell demonstrated significance differences above 0.5 µM.
A99, A2G80, AG73 and EF1m-ELPs and the same trends followed similarly to the area and
perimeter (P < 0.005) (Figure 3C).
56

Figure 18. Dose-dependent cell spreading on LELP coated plates. A99, A2G80, AG73 and EF1m fused
ELPs or I30 only were coated in non-tissue culture treated 96-well plates at 37 °C for 1 hour and 2x10
3

HDFs were seeded and incubated at 37 °C for 2 hours in blocking media. A) The average cell area in µm
2
.
B) The average perimeter length around the cell in µm. C) The maximum axis length spanning the longest
ends in µm. The coated LELPs begin to show a statistically significant increase in bioactivity at 0.5 µM
compared to I30 only (P < 0.005). All the parameters steadily rise as the LELPs concentration increase
except A2G80-ELP coated plates lose their ability to promote cell spreading at 10 µM. Error bars indicate
the 95% confidence interval.

While these quantifiable parameters revealed similar trends, the morphology of the HDFs
generally differed among the coated LELPs. Figure 19) shows representative brightfield
micrograph images at 40x for each group. A99-ELP coated HDFs spreads out well, while A2G80-
ELP coated HDFs have many processes that extend from the body of the cell (Figure 19A and
57

19B). AG73-ELP coated HDFs spreads out greatly as well but have a distinctly different
morphology compared to A99-ELP coated HDFs (Figure 19C). AG73-ELP coated HDFs have a
more circular structure while A99-ELP coated HDFs stretch out in a long axis. EF1m-ELP coated
HDFs behave similarly to A2G80-ELPs coated HDFs and also have processes; however, their thin
processes extend much farther (Figure 19D). While I30 control dishes contribute to laminin non-
specific binding of HDFs at high concentrations, those cells remain round in shape when plated at
low seeding densities (Figure 19E).
Confocal laser scanning images demonstrate that A99-ELP coated plates with HDFs have
a higher spreading factor compared to other LELPs (Figure 20A). The vinculin staining pattern
for HDFs on A2G80-ELP coated plates shows that they are predominantly found facing the
perimeter of the cell (Figure 20B). A2G80-ELP promote small processes that extend from the
main body of the cell. HDFs on AG73-ELP coated plates have a unique F-actin arrangement
consisting of a circular cell shape with ruffles at the edges (Figure 20C). AG73-ELP are the only
LELPs to promote this behavior. EF1m-ELP coated plates show that when HDFs are bound
attached, the EF1m peptide promotes thin, elongated processes (Figure 20D). The HDFs tend
towards an elongated morphology as well. Figure 20E shows the effect of ELP I30 with only
HDFs attached. While there is non-laminin mediated cell attachment at higher concentrations,
when plated at these low seeding densities, HDFs retain a rounded morphology.

58

Figure 19. LELP fusions demonstrate distinct cell adhesion and morphology. 1 µM of A99, A2G80,
AG73, EF1m ELP fusions or I30 only were coated on 96-well non tissue culture treated plates and seeded
with 5x10
3
HDFs in 0.1% BSA and basic DMEM (no serum) for 2 hours at 37 °C. The cell adhesion and
morphology were examined by brightfield microscopy. A) Coated A99-ELP plates provide excellent cell
attachment and very large (area) cell spreading. B) A2G80-ELP coated plates spread less than A99-ELP
and have very fine neuronal-like processes. C) Coated AG73-ELP wells demonstrate a round ruffling
pattern with a large surface area. D) EF1m-ELP coated wells provide adequate cell adhesion and has
elongated processes at the tips. E) I30 demonstrates minimal cell attachment. Images were modified with
the ‘auto tone’ function of Adobe Photoshop to consistently increase the contrast of the cell and background
image. White bar represents 100 µm.

59

Figure 20: Vinculin and F-actin arrangement during cell attachment and spreading. I30 and the
LELPs were prepared similarly as figure 4, however into black 96-well, clear bottom, non-treated plates.
Immunostaining of vinculin (green), F-actin (red) and the nuclei (blue) was performed on plated HDFs to
show the protein distribution. A) Coated A99-ELPs demonstrates great cell spreading by the strong focal
adhesions (white arrows) and clear F-actin filament staining. B) Plates coated with A2G80-ELPs show
multiple processes that protrude from the body (white arrows). C) Coated AG73-ELPs have a more
common circular shape and form ruffles at the edges (white arrows). D) Plated EF1m-ELPs form processes
similar to A2G80-ELPs, however, they are much longer (white arrows). E) I30 only, while does have non-
specific cell adhesion activity, does not spread well. White bar represents 20 µm.

3.4.5. Cell attachment activity with mixed LELPs
To directly explore synergy between different members of the LELP library a total peptide
concentration was fixed for dish coating and 1:1 mixtures between pairs of peptides were
compared to dishes coated with a single peptide. In this assay, synergy would be expected if a
mixture promoted cell attachment and spreading at a level greater than for each peptide alone.
60

Mixtures of A99, A2G80, AG73 or EF1m-ELPs were coated in a 1:1 ratio at 0.25 or 0.5 µM each,
and the number of HDFs that attached after a 2 h incubation at 37 °C was evaluated. At low coating
concentrations of 0.25 µM, there was little cell adhesion activity with most of the mixed LELPs
(Figure 21A). Mixture of AG73:EF1m-ELP and EF1m:A2G80-ELP produce little cell attachment,
which appeared on the level of non-specific adhesion. AG73:A2G80 and A99:EF1m-ELPs coated
at 0.25 µM show some HDF adhesion; furthermore, this expanded at a coating concentration of
0.5 µM. Surprisingly, dishes coated with A99:A2G80-ELPs mixed 1:1 at 0.25 µM have a
significant difference compared to all the other combinations and a significant mean increase of
54 HDFs attached to the plate with A99:A2G80-ELPs compared to only 18 in the A99:AG73-ELP
coated group (P = 0.005). When the mixture concentration increases to 0.5 µM each (Figure 21B),
all mixtures reach their maximum bioactivity except for AG73:EF1m, which performs worse than
other combinations. While A99:A2G80 at 0.25 µM each provides excellent cell attachment activity,
there is a significant difference compared to that of its 0.5 µM counterpart (P = 0.009). Figure
21C is a brightfield image comparing the difference and similarities of cell attachment activity of
coated A99:A2G80 and A99:AG73-ELPs at 0.25 and 0.5 µM.
61

Figure 21. Cell attachment activity of mixed ratios. A99:A2G80, A99:AG73, A99:EF1m, AG73:A2G80,
EF1m:A2G80 and AG73:EF1m fused to ELPs were coated in non-tissue culture treated 96-well plates and
cell adhesion activity was assessed. A) Micrograph images of 0.25 or 0.5 µM, 1:1 ratio of A99:A2G80 and
A99:AG73 coated plates with 5x10
3
HDFs attached after 2 h at 37 °C. B and C) The average number of
HDFs attached when 0.25 or 0.5 µM equal ratios of the LELPs are coated. ** P < 0.005. White bar
represents 200 µm in images. Error bars are standard deviation.

62

When comparing between A99 and A2G80 coated at 0.5 µM, at a seeding density of 2x10
4

HDFs for 1h at 37 °C, A99:A2G80-ELP coated at 0.25 µM each has significantly more cells
attached with a mean of 206, 168 and 324 cells per field respectively (P < 0.0001) (Figure 22A).
At 1 µM, A99 and A2G80-ELPs also has a significantly fewer cells attached compared to dishes
coated with A99:A2G80-ELPs (P < 0.0001). Moreover, A99:A2G80-ELP coated plates also
demonstrated better cell spreading with a mean area compared to A99 or A2G80-ELP coated
dishes. The maximum axis length and perimeter of cells attached on A99:A2G80-ELP coated
plates were also significantly different compared to cells plated on A99 or A2G80-ELP alone (P
< 0.0001) (Figure 22B). Interestingly, when HDFs are plated on A99:A2G80-ELPs, they
demonstrate a qualitative mixture of both morphologies (Figure 22C) approximately 10-20% of
the time. The HDFs tend to show strong focal adhesion points at the far edges of the cell yet do
not always have tight F-actin filament staining opposite of A99. Additionally, there is strong
overlap at the edges where vinculin and F-actin colocalize. Figure 22D includes comparative
images of HDFs on A99 and A2G80-ELP coated plates that shows the two distinct phenotype that
Figure 22C shares. Based on the observation that cell attachment and spreading were both
enhanced for the combination of A99 and A2G80-ELP peptides compared to single LELP coated
dishes, it appears these two formulations promote a synergistic effect on morphology.
63

64

Figure 22: Synergistic cell adhesion and spreading activity of A99:A2G80-ELP. A99:A2G80-ELP
coated plates at 0.25 µM were compared with coated A99 or A2G80-ELP at 0.5 µM. A) When 2x10
4
HDFs
were seeded onto the coated plates, A99:A2G80-ELPs shows stronger cell attachment than A99 or A2G80-
ELP (P < 0.0001). B) The mean area (µm
2
), maximum axis length (µm) and perimeter (µm) was compared
between 2x10
3
HDFs coated on A99, A2G80, and A99:A2G80-ELP plates. A99:A2G80-ELP at 0.25 µM
shows statistical significance among all the parameters compared to A99 or A2G80-ELP at 0.5 µM (P <
0.0001). C) 5x10
3
HDFs on A99:A2G80-ELP coated plates have shared morphology between A99 and
A2G80-ELPs. The white arrows point to the strong vinculin focal adhesion points and the F-actin fiber
formation does not stretch from the focal adhesion points. Moreover, there is strong vinculin and F-actin
co-staining at the edge of the cell. D) HDFs on A99 or A2G80-ELP coated plates for staining comparison.
Error bars for number of cells attached are standard deviation while area, maximum axis length and
perimeter is 95% confidence interval.

3.4.6. Inhibition of HDF adhesion and spreading
To explore the specificity of HDF attachment and spreading, cells were subjected to EDTA or
heparin sulfate to determine if this would interfere with their ability to adhere and spread on LELP
coated surfaces (Table 5). When coated, the entire LELP library saw a reduction in HDF cell
spreading when 5 mM EDTA was added; however, some cell adhesion remained whereby cells
with a rounded morphology were observed. (Figure 23). Unlike the other laminin peptides which
binds to integrins, the AG73 peptide is known to bind to cell-surface syndecans. With the addition
of EDTA, HDFs bound to AG73-ELP coated plates continued to attach but did lose their usual
ruffling morphology; however, when 5 mM of EDTA and 10 µg/ml of heparin sulfate were mixed,
HDFs seeded onto AG73-ELPs coated wells saw a reduction in both cell adhesion and spreading.

65

Table 5. Biological activity of LELPs
PC-12 HDF
adhesion/spreading
inhibition
2

Polypeptide
1
Adhesion Neurite outgrowth
I30 - - NA
A99-ELP ++ + EDTA
A2G80-ELP + + EDTA
AG73-ELP ++ + EDTA/HS
EF1m-ELP + - EDTA
A99:A2G80 ++ + EDTA
1
LELPs assayed at a total of 0.5 µM
2
0.5 mM EDTA or 10 µg/ml heparin sulfate preincubated with HDFs
- Indicates no activity.

+ Indicates moderate activity compared to control (I30)
++ Indicates strong activity compared to control (I30)

3.4.7. Neurite outgrowth of PC-12 cells on LELP coated plates
Having demonstrated enhanced attachment and cell spreading induced by the LELP coating, an
additional functional assay was performed using the neuroblastic potential of the PC-12 rat-derived
cell line. PC-12 cells grown on 0.5 µM LELP coated plates demonstrate neurite outgrowth post 72
to 96 h after seeding (Table 5). Specifically, coated A99, A2G80 and AG73-ELP plates had neurite
outgrowth in serum-free media and without collagen. In contrast, EF1m-ELP coated plates had
very few neurites coming from the body of the PC-12 cells. I30 had no effect on neurite outgrowth
at 0.5 µM. A99:A2G80 mixture (0.25 µM each) also demonstrated PC-12 cell adhesion and neurite
outgrowth (Figure 24).

66

Figure 23. Brightfield micrograph images of 2x10
4
HDFs plated on LELP coated plates. The first
column represents no treatment. In the second column, 5mM of EDTA was pretreated with the cells for 15
min. The third column was treated with 10 µg/ml of heparan sulfate (HS) for 15 min, while the last column
was treated with both EDTA and HS for 15 min. Qualitative assessment show a decrease in HDF
attachment/spreading with EDTA among all constructs, however, AG73-ELP is greater affected when there
is an addition of HS with EDTA due to the blockade of syndecan binding.

67

Figure 24. Neurite outgrowth of PC-12 cell coated on LELPs. 5x10
3
cells/well were primed with 100
ng/ml of β-NGF in RPMI media for 24 h. Then, the cells were plated in 96-well plates in advanced
DMEM/F-12 media supplemented with 100 ng/ml of β-NGF and 20 nM of progesterone for 96 h. Cells
primarily with neurites greater than twice the body size is considered to have neurite outgrowth. White bar
represents 100 µm.

68

3.5. Discussion
The concept of biomaterials that are compatible with cells by mimicking the ECM has evolved
with advancements in technologies. At a minimum, biomaterials that promote cell adhesion and
spreading remain essential. According to literature, Heilshorn, Tirrell and colleagues were the first
to engineer a bioactive peptide with an ELP to promote cell adhesion for application in vascular
grafts (Heilshorn, DiZio et al. 2003). They fused the CS5 domain of fibronectin to
[(VPGIG)2VPGKG(VPGIG2)]4 or (VPGIG)25 and repeated the sequence to display multiple CS5
domains interspersed with the ELPs. The group demonstrated excellent cell adhesion of human
umbilical vascular endothelial cells (HUVEC) onto their artificial extracellular matrix and
measured the number of remaining cells attached after subjected to sheer stress. Their research
focused on the biocompatibility of their material for implantation, while our focus is the relative
strength of each peptide in relation to cell spreading. Their approach first showed ELPs as
promising scaffolds, which opened the path for ELP-based ECM-mimetics.
This study expands on our previous collaborative research and introduces 3 additional bioactive
laminin peptides fused to ELPs (Truong, Hamada et al. 2020). A2G80 (VQLRNGFPYFSY) of the
laminin α2 LG4 domain binds to α-dystroglycans, AG73 (RKRLQVQLSIRT) and EF1m
(LQLQEGRLHFMFD) both from the laminin α1 LG4 domain binds to syndecans or integrin α2β1,
respectively. While several laminin peptides have been studied as ECM-mimetics, A2G80 and
EF1m have not been investigated with ELPs as the backbone. Kakinoki and Yamaoka were the
first to design AG73-(VPGIG)30 and showed its thermo-responsiveness promotes adsorption onto
poly(lactic acid) (Kakinoki and Yamaoka 2014). They demonstrated excellent neurite outgrowth
of PC-12 undifferentiated cells which agrees with our results. While their AG73 sequence is the
same as ours, they also included a His-tag for affinity column purification whereas ours is linked
69

to ELP alone. Their Tt in PBS is lower than ours perhaps due to the differences in the linker and
c-terminal amino acid sequences. In table 3, the measured MALDI MWs for A2G80-ELP, AG73-
ELP and EF1m-ELP are slightly greater than the expected MWs. We think that this small
difference might be due to adducts coming from our buffers or the MALDI matrix. ELPs are an
excellent backbone since they are also derived from the ECM, have some cell adhesion bioactivity,
and provide a method for concentrating peptides quickly on surfaces. We have previously
characterized A99-ELP; however, we include it here both for comparison and because the amino
acid sequence linker has been changed due to a different cloning strategy. These bioactive peptides
were fused to the N-terminus of I30 (ELP) using a series of ‘cassettes’ that are PRe-RDL
compatible (McDaniel, Mackay et al. 2010). PRe-RDL designed plasmids allow for seamless
integration without extraneous nucleotides or expressed amino acids. Additionally, this design
allows future iterations of LELPs to be coupled at the C-terminus (bi-headed) or added in
sequential order at the N or C-terminus (multi repeats or multi-faceted). Furthermore, the I30 ELP
sequence can be replaced with another ELP scaffolding sequence such as a shorter sequence, less
hydrophobic sequence, a di-block copolymer or one that contains reactive side chains for covalent
crosslinking. For example, Mie, Mizushima and Kobatake demonstrated that using different ELP
backbones with the RGD sequence act as a temperature-responsive surface for human lung cells
(A549) which could mobilize detached cell sheets (Mie, Mizushima et al. 2008). While many
materials have been tested to promote cell spreading and activity, ELPs are useful scaffolds to
display the laminin peptides because the ELP-mediated phase separation allows purification in
large quantities compared to chemical synthesis and costly chromatography purification. It is
useful to note that while we used the ELPs thermo-responsiveness to purify the LELPs, Heilshorn,
Yamaoka and Kobatake used affinity chromatography instead.
70

The LCST of these fusions are largely dependent on hydrophobicity. The EF1m peptide
sequence is the most hydrophobic, composed of 46% hydrophobic uncharged residues with a
relative hydrophobicity value of 35.91 (ThermoFisher Scientific Peptide Analyzing Tool). We
chose to deviate from the original EF1 sequence (DYATLQLQEGRLHFMFDLG) despite having
greater cell adhesion activity because of its highly hydrophobic amino acid sequence and instead
selected EF1m which is the minimum sequence demonstrating bioactivity. The Tt of EF1m is 12.5
C at 10 µM above which it develops into a gel-like substance and falls out of solution. This is
consistent with its interesting OD profile as temperature increase (from 12.5 C yet falls as soon
as the temperature increases to 25.7 C, Figure 16E and Figure 25). To address the very low Tt
for EF1m-ELP, it may be useful in the future to use a less hydrophobic ELP backbone such as the
valine library (VPGVG) to increase the Tt, which would allow for better handling and usage in the
laboratory. A99-ELP has a higher Tt than I30 likely because the two polar amino acids, D and R
at a pH of 7.4 increases the solubility having a low relative hydrophobicity value of 19.88.
A2G80’s measured Tt at 10 µM is also low at 15.7 C. The amino acid sequence contains
approximately 42% hydrophobic amino acids with a relative hydrophobicity value of 33.09 and
only one polar residue, R. While the sequence is only 12 amino acids in length, it also has a large
influence on the Tt compared to I30 without any fusion sequence. AG73’s Tt at 10 µM was
measured to be 20.5 C and contained only 33% hydrophobic residues L, V, L, I. The composition
contains 33% polar amino acids with a relative hydrophobicity value of 26.99. The amino acid
composition of the laminin peptides has a large effect on the Tt of the I30 ELP due to the
hydrophobicity and protonation status of polar amino acid residues. Additionally, the relative
hydrophobicity values have an inverse relationship with the Tt. We previously published our rapid
coating method with A99-ELP using a temperature greater than 60 C; however, based on the Tt
71

as a function of concentration and the amino acid’s hydrophobicity for the LELPs, we decided to
reduce this temperature to 37 C. This coating step results in adequate polypeptide coating in the
96-well plates (Truong, Hamada et al. 2020). It is important for the phase transition to occur for
better coating of the polypeptide to the plate (Truong, Hamada et al. 2020).

Figure 25. Purified I30 only and EF1m-ELP in Tm microcuvettes above T t. 50 µM each were incubated
between 37 – 40 C and observations show that I30 forms a cloudy yet uniform coacervate within the entire
liquid space, yet EF1m-ELPs come together quite tightly to form a gel-like substance that collapses over
time due to gravity. This matches the data from S3 graph E, where the OD decreases over time/temperature
from supplementary figure S2. Note that the other laminin peptide-ELP constructs behave similarly to I30
with a uniform cloudy appearance.

HDF cell adhesion and spreading for each construct coated shows they each produce
distinct morphologies. A99-ELP when coated as shown previously, has demonstrated great cell
attachment and spreading, as evident by the F-actin fibers. A2G80-ELP coated plates typically
72

show HDFs forming many neuron-like processes at its ends. AG73-ELP coated plates resulted in
HDFs forming a circular ruffle morphology, while EF1m-ELPs resulted in thin processes at their
ends. While these morphologies were distinct for each LELP, there was some overlap depending
on where the micrographs were imaged. The different laminin peptide constructs all provide
maximal cell adhesion activity beginning at 0.5 µM and the spreading activity based on the mean
cell area, perimeter and max axis length significantly increases as well. This trend continues for
A99, AG73 and EF1m-ELP constructs, however, A2G80-ELP coated at 10 µM significantly loses
its ability to promote cell adhesion and spreading. This finding is interesting because A2G80-ELP
produced the best synergy when mixed with another peptide at low concentrations. The rationale
for preparing a mixture of LELPs was to determine if any combination would provide an additive
or synergistic effect. Interestingly most of the LELP constructs did not have a synergistic effect,
except for A99:A2G80-ELPs. While it is unclear why 10 µM A2G80 prevents cell adhesion and
spreading compared to the other constructs, it does seem to be peptide sequence and scaffold
specific and should be further evaluated for cell toxicity. Since it is the first and only sequence
specific peptide that was found to bind to α-dystroglycan, follow up studies should be considered
(Suzuki, Hozumi et al. 2010). At 0.25 µM equimolar ratios, there was very little cell adhesion;
however, these results demonstrate the importance of A99-ELP (which includes the RGD amino
acid sequence) and show the A99:A2G80-ELP combination has similar bioactivity at half the
concentration of other treatments. An interesting finding regarding the cell adhesion and spreading
inhibition assay is that while EDTA prevents integrin binding, the cells remain attached to the
wells, albeit with a rounded, non-spread morphology. It is also interesting that both EDTA and
heparin sulfate are required to maximally inhibit the cells from AG73-ELP coated plates.
Moreover, when coating A99-ELP, A2G80-ELP and AG73-ELP at 0.5 µM, PC-12 cells, which
73

are usually floating in clusters or loosely attached to the plates, became adherent and capable of
stimulated neurite outgrowth.

3.6. Summary
This chapter demonstrates the versatility of these LELPs and their ability to be used individually
or in combination with other LELPs. They provide great biological activity on a format that is easy
to use. LELPs may have future applications as ECM-mimetics for cell therapies and tissue
engineering.

3.7. Acknowledgements
A.T.T. thanks Dr. Taojian Tu for his insightful comments and support. This work was made
possible by the Japan Society for the Promotion of Science (PE18045) to A.T.T., the United States
National Institutes of Health RO1 GM114839 to J.A.M., R01 EY030141-A1 to J.A.M., P30
EY029220 to the USC Ophthalmology Core Grant in Vision Research, P30 CA014089 to the USC
Norris Comprehensive Cancer Center, the Gavin S. Herbert Endowed Chair of Pharmaceutical
Sciences, Dr. Junji Watanabe from the Translational Research Laboratory and Dr. Alireza
Abdolvahabi from the Mass Spectrometry Core at USC School of Pharmacy.

74

CHAPTER 4: TEMPERATURE-RESPONSIVE ESTROGEN
RECEPTOR FUSIONS SELF-ASSEMBLE INTRACELLULARLY

4.1 Abstract
Protein design and engineering is becoming increasingly popular for generating proteins with
useful properties and is an effective method to modulate a protein’s function. Recent focus on
phase separation have provided a new framework for our understanding of the functional role of
microdomains. Here, we describe the development of nuclear hormone estrogen receptor α (ERα)
fused to elastin-like polypeptides (ELPs) to form a fusion protein (V96-ERα) with remarkable
properties. ELPs are thermally responsive biopolymers that self-assemble and induce phase
separation of protein-rich microdomains within living cells. V96-ERα fusions have been
demonstrated to form clusters both in the cytosol and nuclei above its transition temperature (Tt).
When stimulated with estradiol, V96-ERα puncta formation is found more often in the cytoplasm
suggesting there is a reduction in nuclear translocation. The design of V96-ERα may enable the
study of spatiotemporal ERα intracellular signaling.

4.2 Introduction
Recombinant DNA technology has enabled us to develop novel synthetic proteins with multi-
functional properties. This is achieved by genetically incorporating two or more proteins or their
domains, resulting in a fusion that produces multiple distinct functions derived from their
individual moieties. Over the last several decades, this strategy has been used to harness the power
of various functional protein domains.(Markland, Roberts et al. 1991, Hassouneh, MacEwan et al.
75

2012, Levin, Golding et al. 2015, Yu, Liu et al. 2015, Ren, Wen et al. 2021) For example, popular
biological applications have incorporated peptide or affinity tags for protein purification, they have
added fluorescent proteins to visualize and track fusions within living cells and have even have
constructed artificial fusion proteins that act as intracellular switches.(Smith and Johnson 1988,
Van Dyke, Sirito et al. 1992, Matz, Fradkov et al. 1999, Wahlfors, Loimas et al. 2001, Terpe 2003,
Pastuszka, Okamoto et al. 2014, Li, Tyrpak et al. 2018, Tyrpak, Wang et al. 2020) The endless
possibilities of fusion proteins enable opportunities to explore and discover new protein multi-
functions that may be useful in biological and biomedical research.
Cells are comprised of a well-defined, highly compartmentalized mixture of biomolecules
such as proteins, lipids, carbohydrates or nucleic acids. These compartments specialize in diverse
functions and are arranged in a manner that modulates biochemical reactions or biological
processes that support cellular activities. While membrane-bound compartments are well
understood and described extensively in literature, biomolecular condensates or membrane-less
compartments and their biological function are still being investigated.(McSwiggen, Mir et al.
2019) Some have suggested that self-assembly and phase separation of molecules allows
components to be rapidly concentrated and drive processes ranging from gene expression to cell
division and more.(Hyman, Weber et al. 2014, Brangwynne, Tompa et al. 2015, McSwiggen, Mir
et al. 2019) Some of these molecules include intrinsically disordered proteins (IDPs) which have
previously been demonstrated to facilitate a variety of biological processes.(Gomes and Shorter
2019) Since these are distinctly different mechanisms to control specific cellular activities, the
dynamics may be a result of these spatiotemporal controls of the condensates.
Nuclear hormone receptors are ligand-dependent transcriptional regulators that modulate
gene expression for an assortment of biological processes, ranging from cell proliferation to
76

metabolism or inflammation. Estrogen receptor α (ERα) is a member of this family and has
demonstrated as an important transcription factor that mediates many biological responses relating
to reproduction, cardiovascular health, skeletal homeostasis and central nervous system function.
While ERα was historically known for its slower genomic transcriptional actions, the present
understanding has introduced and highlighted rapid, nongenomic signaling in the cytoplasm and
at the plasma membrane surface.(Moriarty, Kim et al. 2006, Arnal, Lenfant et al. 2017, Barton,
Filardo et al. 2018) More recently, intense research to describe these rapid responses and increase
our understanding of their multifaceted biological roles have been a major focus area.(Lu and
Herndon 2017) Creating and exploring novel tools that promote local clustering such as the
naturally occurring membrane-less compartments may help delineate and provide valuable
information on other convoluted ER-mediated rapid signaling in the cytoplasm.
Elastin-like polypeptide (ELPs) are a class of recombinant polymers that are thermally
responsive and rapidly self-assembly forming microdomains within a cell. The clustering is
triggered above a lower critical solution temperature (LCST), also known as the transition
temperature (Tt), which initiates the onset of phase separation. Our research group has recently
reported that proteins fused to ELPs become thermally responsive while retaining their normal
biological activity. Below the Tt, the protein-ELP fusions behave normally; however, as the
temperature is increased above the Tt, the ELP moieties rapidly phase separate, concentrate
attached biomolecules, which alters their activity. For example, ELPs have previously been fused
to clathrin light chain and, when triggered to phase separate, inhibit clathrin-mediated endocytosis
by inhibition of the triskelion assembly.(Pastuszka, Okamoto et al. 2014) Additionally, when these
ELPs were fused to EGFR, microdomain formation promoted MAPK signaling in a pulsatory and
reversible manner.(Li, Tyrpak et al. 2018) This was postulated to promote oligomerization and
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autophosphorylation of their intracellular domains. By fusing ELPs to specific proteins with
significant pathophysiological relevance, this may enable us to use this novel method to promote
phase separation within live cells to mimic biomolecular condensates and better understand their
relevance as it relates to dysregulation and disease.
An ELP moiety called V96-ELP was fused to ERα (called V96-ERα) to induce phase
separation and to study their behavior. V96-ERα demonstrated thermo responsiveness forming
puncta within living cells at physiologically relevant temperatures, while remained dispersed in
the cell below its predicted transition temperature. When V96-ERα was treated with estradiol (E2)
in MCF7 cells, the fusions were more likely to be found in the cytoplasm at physiological
temperatures compared to lower temperatures. This fusion protein concentrates within cells,
mimicking biomolecular condensates and may become a useful tool to expand our knowledge of
ERα plasma membrane and cytoplasmic rapid signaling in a spatiotemporal manner.

4.3 Material and methods
4.3.1. Plasmid construction
An ERα mammalian expression vector, pcDNA-HA-ER WT (Addgene plasmid 49498), was
modified by traditional molecular cloning. Briefly, an oligomer with a sequence of 5’ -
gatcctcggatcGATATCag- 3’ (sense) and 3’ -gagcctagCTATAGtcctag- 5’ (antisense) containing
an EcoRV blunt-end restriction endonuclease site was inserted into the plasmid using BamHI-HF
(R3136S, New England Biolabs, Ipswich, MA). V96-ELP plasmid (Addgene plasmid 68392)
previously generated by our laboratory by recursive directional ligation was double-digested with
NdeI and AcuI (R0111S and R0641S), and the fragment excised and purified from a 1% agarose
78

gel with a QIAquick Gel Extraction kit (28706, Qiagen, Hilden, Germany). A blunt-end reaction
with Klenow (M0210S) was performed on the fragment and subsequently purified using a
QIAprep Spin Miniprep kit (27106). The pcDNA-HA-ER WT vector containing the
oligonucleotide insert was linearized with EcoRV-HF (R3195S) and the 5’ phosphate group
removed using Antarctic Phosphatase (M0289S). The linearized plasmid was then purified from a
1% agarose gel and a 1:3 vector to insert molar ratio was used to ligate (T4 DNA ligase, M0202T)
the two plasmid DNA strands overnight at 16 C in a thermocycler. Subsequently, the ligation
reaction was transformed in TOP10 competent cells (C404003, Thermo Fisher Scientific, Waltham,
MA) according to the manufacture’s protocol, plated on carbenicillin-containing agar plates and
incubated at 37 C overnight. Colonies were screened by performing a diagnostic digest with KpnI-
HF (R3142S) and XbaI (R0145S) and verified by Sanger sequencing (Retrogen Sequencing, San
Diego, CA). The resulting fusion is labeled as V96-ERα.
4.3.2. Cell culture and DNA plasmid transfection
HEK-293T and MCF7 cells (CRL-3216 and HTB-22, ATCC, Manassas, VA) were maintained in
DMEM (11965092, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS (35-
011-CV, Corning Inc., Corning, New York) at 37 C in a 5% CO2 humidified tissue culture
incubator. 2.5x10
4
cells were seeded onto poly-D-lysine coated glass coverslips in a 12-well dish
with phenol red free DMEM (21063029, Thermo Fisher Scientific, Waltham, MA) supplemented
with 5% charcoal-stripped FBS (CS) (A3382101, Thermo Fisher Scientific, Waltham, MA) and
incubated at 37 C overnight. The following day, cells were transfected with Lipofectamine 3000
according to the manufacture’s protocol with phenol red free Opti-MEM (L3000008 and 11058021,
Thermo Fisher Scientific, Waltham, MA) and incubated for an additional 48 h at 32 C. Opti-
MEM was replaced with phenol red free CS DMEM 6 to 8 h post transfection.
79

4.3.3. Immunoblot detection
HEK-293T cells were transfected with both the wild-type and V96-ERα fusions in a 6-well dish
and collected using RIPA buffer containing protease inhibitor (78430, Thermo Fisher Scientific,
Waltham, MA). Cell lysates were frozen at -80 C then briefly sonicated on ice (Branson Sonifier
150, St. Louis, MO). The soluble fraction was collected, and an estimated 20 µg of total protein
were resolved by SDS-PAGE. The protein was transferred at 300 mA for 90 min onto a pre-
activated Immobilon-P PVDF membrane (IPVH00010, Millipore Sigma, Burlington, MA) and
block in 5% non-fat milk in TBS-T (sc-2325, Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h.
Rabbit anti-HA antibodies (C29F4, Cell Signaling Technology, Danvers, MA) was added in a
1:1000 dilution and incubated at 4 C overnight. The following day, the membrane was washed
three times with TBS-T for 10 min each and a 1:5000 dilution of anti-rabbit HRP-linked secondary
in milk was added at room temperature for an additional h. After an additional three washes with
TBS-T, proteins on the membrane were detected with the addition of chemiluminescent substrates
(1856135, Thermo Fisher Scientific, Waltham, MA) and captured on an iBright imaging system
(FL1000, Thermo Fisher Scientific, Waltham, MA).
4.3.4. Immunofluorescence and confocal microscopy
Fixed slides were analyzed using a LSM880 laser scanning confocal microscope with Airyscan
(Carl Zeiss, Jena, Germany). Briefly to prepare the slides, coverslips were fixed by incubating
them in 4% paraformaldehyde (PFA) for 15 min, then washed in 50 mM of ammonium chloride
for 5 min. The coverslips were then briefly washed three times with PBS for 5 min and
permeabilized with 0.05% triton X-100 for 10 min. After, another three washes with PBS for 5
min each was performed and blocked with 1% BSA in PBS for 1 h. 1:100 rabbit anti-HA antibody
80

in 1% BSA was incubated at 4 C overnight and washed with PBS three times for 5 min the
following day. Next, 1:100 anti-rabbit Alexa Fluor 488 (212106, Thermo Fisher Scientific,
Waltham, MA) in 1% BSA was incubated on the coverslips for 1 h at room temperature followed
by a quick 5 min DAPI staining. The coverslips were thoroughly washed with PBS an additional
three times and mounted on slides with Prolong Antifade (P36934, Thermo Fisher Scientific,
Waltham, MA).
4.3.5. Subcellular localization
MCF7 cells were plated on coverslips with 5% CS FBS in DMEM and transiently transfected with
V96-ERα as previously described. Approximately 48 h later, the media was replaced with fresh
ice-cold 5% CS FBS in DMEM and placed at 4 C for 30 min. The media was replaced with
prewarmed DMEM (10, 20, 30 or 37 C) and transferred onto a heating dry bath (IC25, Tory Pines
Scientific, Carlsbad, CA) for an additional 30 min at the above indicated temperatures. The cells
were then treated with media containing 10 nM of 17 -estradiol (10006315, Cayman Chemical,
Ann Arbor, MI) or DMSO (D12345, Thermo Fisher Scientific, Waltham, MA) as a negative
control and incubated for another 30 min. Cells were immediately fix with room temperature 4%
PFA as described above.
4.3.6. Data and statistical analysis
Localization of V96-ERα was repeated three times and the data are presented as the mean ± SD. 4
confocal images with 1 or more transfected cell(s) representing the general population were
acquired at different temperatures. These images were analyzed by CellProfiler to determine if
V96-ERα was found predominantly in the cytoplasm or nucleus. Briefly, the pipeline first
determined the nucleus in the DAPI channel and measured the integrated density in the region of
81

interest (ROI). After, the total cell was identified in the second channel containing V96-ERα and
the integrated density of the nucleus was subtracted by the total cell resulting in the integrated
density remaining in the cytoplasm. A value greater than 1 represents more signal in the cytoplasm,
while a value less than one suggested more signal in the nucleus. Coacervation of the fusion protein
was determined by blinding the research to the transition temperatures and showing the confocal
images in a random order. The 48 confocal images had their filenames randomized to numerical
values using a python script similarly to previously described.(Tyrpak, Wang et al. 2020) The
transitioned-microdomain formation data was compared against the integrated data ratios at
different temperatures and plotted with PRISM (GraphPad 6, San Diego, CA). A multiple student
t-test using the Holm-Sidak correction method was performed for each temperature comparing
transitioned and nuclear staining.

4.4. Results
4.4.1. Generation of thermally responsive ELP-ERα
V96-ELP was fused to full length ERα using traditional cloning (Figure 26A) and the translated
amino acid sequences of V96-ELP, ER-WT and the fusion V96-ERα are shown on Table 6. A
diagnostic digest of the ER-WT and V96-ERα demonstrated an appropriate size of 1200 and 2640
base pairs respectively (Figure 26B). Additionally, the expected molecular weight of 105 kDa for
V96-ERα fusion was confirmed by transient transfection of HEK-293T cells and immunoblot
analysis (Figure 26C). ELPs are synthetic biopolymers with a pentameric amino acid repeat
consisting of (Val-Pro-Gly-Xaa-Gly)n, where Xaa represents any guest residue and n is the number
of repeats. ELPs phase separate and form microdomains in cells when heated above its Tt and this
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Tt can be genetically tuned to a specific temperature by switching the guest residue or altering ELP
length. A schematic illustration of V96-ERα assembly is shown (Figure 26D). V96-ELP was fused
to the N-terminal domain to prevent impediments in ERα DNA binding domain. The fusion
construct was successfully expressed in HEK-293T cells and microdomain formation was
confirmed at 37 C by confocal microscopy. The expected molecular weights of 67 and 105 kDa
for ER-WT and V96-ERα was confirmed by immunoblot analysis.

Table 6. Amino acid sequence of the plasmids for chapter 4
Nomenclature Amino Acid Sequence
1
Expected Molecular Weight (kDa)
2

ER-WT HA-ER 67.2
V96-ELP G(VPGVG)96Y 39.5
V96-ERα G(VPGVG)96-HA-ER 107.3
1
Complete amino acid sequence open reading frame available in supplemental section
2
Predicted molecular weight calculated by ExPASy Compute pI/Mw tool

83

Figure 26. Design and illustration of proposed ELP-ERα assembly and confirmation of expression.
A) Gene encoding for V96-ELP is fused to the N terminus of full length ERα including a HA tag in between
for indirect immunostaining. B) Agarose gel of a diagnostic restriction digest 1) ER-WT and 2) V96-ERα,
with band sizes of 1200 and 2640 base pairs respectively. C) Schematic of V96-ERα dispersed around the
cell below the predicted T t yet form microdomains when heated to physiological temperatures. D) HEK-
293T transiently expressing 1) ER-WT, 2) V96-ERα with some cleavage or 3) a negative control with
GAPDH as a loading control.

4.4.2. Temperature-triggered microdomain formation of ELP-ERα
To estimate the assembly temperature of V96-ERα, HEK-293T transfected cells were incubated
at 18, 23 or 37 C for 30 min then fixed as previously described above (Figure 27). At 18 C, V96-
ERα remained completely soluble in the cell. However, at 23 C, while some cells exhibit
microdomain formation, a majority of the cells still demonstrated a diffuse staining pattern
indicating no coacervation. At 37 C, more cells have puncta formation within the cell. This shows
that there is a temperature dependence in microdomain formation of V96-ERα expressing cells.
84

Figure 27: Temperature-triggered phase transition of ELP-ERα fusions. HEK-293T transiently
overexpressing ER-WT or V96-ERα. ER-WT show disperse staining within the nucleus, while V96-ERα
has similar staining pattern at 23 C and below. However, at 37 C, V96-ERα phase separated and formed
microdomains. Green: antibody against HA. Blue: DAPI.

4.4.3. Localization of puncta in the cytoplasm at greater temperatures
V96-ERα fusions are found in the cytoplasm more often when grown in CS FBS DMEM inside
MCF7 cells. ERα ligand, E2, at 10 nM promotes translocation from the cytoplasm to the nucleus.
When V96-ERα transfected MCF7 cells were treated with E2 at 10 C, most of the staining
suggests the fusion protein is found predominantly in the nucleus (Figure 28A). There was a
statistically significant difference in the lack of microdomain formation and increased nuclear
85

staining at 10 C (P < 0.0009) (Figure 28B). At 20 C, cells were observed to have both either
puncta forming or diffuse staining patterns. In addition to both observed phenotypes, V96-ERα
was also found to localize evenly in both the cytoplasmic or nuclear space. This resulted in no
significant difference between microdomain formation or localization (P = 0.0667). Furthermore,
the addition of 10 nM E2 appeared to reduce the Tt closer to 20 C providing more of a
heterologous population than when incubated under starving conditions at 23 C. At 30 C, there
was a significant difference between puncta formation of V96-ERα and its location trending
towards the microdomains being found more often in the cytoplasm (P = 0.0143). At 37 C, V96-
ERα was predominantly found in the cytoplasmic compartment in its transitioned, punctate state.
This was significantly different from the other temperatures (P = 0.006074).

86

Figure 28: Microdomain formation is found more often in the cytoplasm than the nucleus. A) MCF7
cells transiently expressing V96-ERα were stimulated with 10 nM of E2 for 30 min at 4 different
temperatures. E2-mediated dimerization lowers the T t from more than 23 C to 20 C or less. B) An inverse
relationship suggesting that an increase in temperature, which increases punctate formation decreases the
number of V96-ERα staining found in the nucleus.

4.5. Discussion
ERα is one of the most well-studied hormone nuclear receptors where much research has focused
on its role as a transcription factor and co-regulator in the nucleus. However, non-genomic
signaling of ERα has been a major focus in the last two decades to comprehend the rapid cellular
responses too quick to be explained by the slow rate of gene transcription. More recently, phase
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separation of various concentrated membrane-less compartments has demonstrated to promote
localized enhanced biochemistry and cell signaling. Using protein engineering, we fused the
domain of an IDP called V96-ELP from tropo-elastin to the full length ERα and created a synthetic,
thermally responsive protein fusion. This fusion phase separates to form microdomains and may
mimic biomolecular condensates which spatiotemporally regulates other cellular activities and
signaling.
This chapter briefly describes the generation of a fusion called V96-ERα that may be used
as a molecular tool to probe and understand ERα rapid signaling by promoting clustering within
cells. Our group has previously reported that when ELPs were fused to GFP, they presented as
distinct puncta when incubated above its expected Tt; however, below the Tt, they appeared evenly
distributed within the cell. These temperature responsive protein fusions Tt are tightly controlled
through the ELPs amino acid composition and pentameric repeats. They form microdomains and
can essentially act as a ‘switch’ which our lab has demonstrated specific modulation of significant
biological processes. Similar to this behavior, when V96-ERα is below its predicted Tt at 18 C,
the staining pattern resembles free endogenous ERα. Under serum starving conditions at
temperatures near the predicted Tt such as 23 C, there are two populations of cells expressing
V96-ERα, one with a dispersed staining pattern like the wild type and the other showing strong
puncta. This variation in phenotype could be due to differences in transient transfection
efficiencies which would change the total concentration and therefore change the Tt of each cell.
This could be addressed by creating and selecting a stable cell line where all cells express the same
amount of V96-ERα. At physiological temperatures, we observe more cells having a punctate
phenotype when compared to the previous temperatures. It seems that V96-ERα assembles to form
microdomains both in the nucleus and cytoplasm. When verifying the expression of V96-ERα, the
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immunoblot shows the appropriate molecular weight of nearly 107.3 kDa; however, there is some
staining at 65 kDa as well, suggesting that the linker between V96-ELP and ERα has some
susceptibility for cleavage.
Experiments were conducted in MCF7 ERα positive breast cancer cell lines using the same
protocols with the addition of E2. When incubated at 10 C, the addition of 10 nM E2 in MCF7
transfected cells showed most of the staining localized in the nucleus with a scattered pattern. At
20 C, with the addition of 10 nM E2, there were a mixture of cells which had staining both in the
cytoplasm, nucleus or both. More interestingly, there were two population of cells that exhibited a
dispersed, non-transitioned staining pattern or a fully punctate pattern. While we demonstrated that
in HEK-293T cells at 23 C, the predominant phenotype was a distributed staining pattern, this
difference is likely due to the addition of E2. Our group previously demonstrated that by adding
two separate ELP molecules together through a single binding partner, that the transition
temperature could be decreased due to the increase in total ELP size.(Dhandhukia, Brill et al. 2017)
Similarly, we predict that the addition of E2 will also reduce the Tt by promoting monomeric V96-
ERα fusion to form a dimer and doubling its length, thus lowering the original Tt. At 20 C, we
see two populations which may suggest that the Tt of the fusion dimer is below or near 20 C. At
30 C we observe more microdomains within the cell, yet when we reach 37 C, there are more
cells that show the phase transitioned phenotype as GFP-fused ELPs. An interesting observation
is that when V96-ERα is transitioned, they seem to be more concentrated at the nuclear surface or
at the plasma membrane than in the bulk of the cytoplasm. This information may be useful for
understanding the spatial location for rapid signaling that previous literature has described.
ELPs, when fused to proteins and expressed within cells act as a ‘switch’ to alter the
protein’s original function. Rapidly and reversibly switching a biological process in an instant is
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very useful; however, the technology requires the addition or subtraction of heat to induce phase
separation of the fusions. This may produce a heat shock response, so a wild-type control is
essential to ensure the resulting change is due to the ELP and not the change in temperature. V96-
ERα coacervates and forms microdomains at physiological temperatures in the cytoplasm and the
nucleus which acts to concentrate the fusion protein to a specific area within the cell. This approach
may intimidate the membrane-less compartments and may be used as a tool to further interrogate
rapid signaling responses induced by ERα. With this technology as a molecular toolbox, there are
endless numbers of opportunities to create fusions with unique properties to discover new protein
function.

4.6. Summary
This chapter briefly describes the development of a new temperature dependent ERα fusion that
self-assembles to form microdomains in live cells. This phase separation mimics biomolecular
condensates and may have utility in studying rapid ERα signaling.

4.7. Acknowledgments
This research was made possible by the American College of Clinical Pharmacy Research Institute
to A.T.T, NIH RO1 GM114839 to J.A.M., and USC Ming Hsieh Institute to J.A.M.

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CHAPTER 5: CHARACTERIZATION OF FLOTILLIN-1
ELASTIN-LIKE POLYPEPTIDE FUSIONS

5.1. Abstract
Understanding the relationship between a protein and its biological function is the cornerstone for
recognizing any associated human disorders. Advancements in molecular biology has given rise
to ‘protein engineering’ allowing the ability to design and construct new proteins with novel
functions. This enables direct analysis of the contribution of the specific modification made to the
protein and captures the proteins functional state. Flotillins are raft-associated integral membrane
proteins involved in a broad range of physiological processes such as endocytosis, trafficking and
cell signaling; however, their etiology associated with various human diseases remains uncertain.
We modify flotillin-1 to express different elastin-like polypeptide (ELP) fusions and characterize
their behavior. ELPs are a class of thermo-responsive biopolymer that are triggered to form protein
microdomains intracellularly. Our group has previously demonstrated that phase separation alters
the biological function of proteins fused to ELPs and apply the same strategy here. Our three
FLOT1-ELP constructs demonstrate phenotypically different staining patterns correlating to three
different behaviors, temperature-sensitive, -insensitive and constitutively ‘on’. Additionally, these
constructs can colocalize with cholera toxin subunit B, a marker for lipid rafts, and follow their
internalization and sorting. This preliminary report can be used to develop further studies and
enhance our understanding of flotillin biology.

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5.2. Introduction
Proteins are macromolecules found in all cells within our body and are essential components to
nearly every biological process necessary for life. Critical cellular activities from gene expression,
protein synthesis or cell cycle progression to cell signaling or cellular trafficking are tightly
controlled through numerous molecular mechanisms to ensure normal cell function and
stability.(Lew 2001, Lopez-Lastra, Rivas et al. 2005, Segev 2009, Feher 2017, Singh, Miaskowski
et al. 2018) Human disorders are largely caused by improper protein function, and therefore,
exhaustive efforts to elucidate a protein’s structure, function, activity, behavior or spatial location
is instrumental to better evaluating abnormalities within cells. (Wang, Huang et al. 2014, Li, Yang
et al. 2016, Hanna, Guerra-Moreno et al. 2019) The fundamental goal is understanding these
mechanisms, and their roles in disease progression. Exploring a protein’s function has routinely
been tackled through overexpressing or knocking out the protein, mutating its key amino acids
residues, truncating specific domains or conducting biochemical assays for qualitative or
quantitate readouts that allows us to better interpret their behavior. However occasionally,
generally accepted traditional assays have not provided a concise picture due to the complexity of
all the machinery in the cell; therefore, research focused on alternative modes to understand
proteins and their function using protein bioengineering has been explored with great
success.(Chen, Zaro et al. 2013, Yu, Liu et al. 2015, Joshi, Rubart et al. 2019, Ruijgrok, Ghosh et
al. 2021)
Our laboratory has recently developed a technology relevant to our research interests that
modulates the activity of proteins within living cells.(Pastuszka, Janib et al. 2012, Pastuszka,
Okamoto et al. 2014, Li, Tyrpak et al. 2018, Tyrpak, Wang et al. 2020) These cells express fusion
proteins that are controlled by modulating the internal temperature of the cell. The proteins are
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fused to a thermo-responsive biopolymer called elastin-like polypeptides (ELPs) which self-
assembles to a more ordered structure based on temperature. ELPs consist of a pentameric amino
acid sequence repeat of (VPGXG)n, where X can be any guest amino acid residue and n is the
number of repeating units. When heated above a specified temperature, the fusion proteins
coalesce to form protein microdomains called a coacervate, leading to changes in their behavior.
This pre-defined temperature is precisely controlled through the ELP guest residue and the number
of repeats. The more hydrophobic the X guest residue or the longer the number of repeats, results
in a lower phase transition temperature also known as the transition temperature (Tt). This
reversible process has previously been shown to alter the biological activity of these proteins or
their intracellular pathways in an order of minutes. For example, we have demonstrated that an
ELP called V96-ELP with an amino acid sequence of (VPGVG)96, when fused to clathrin light
chain, manipulated internalization of angiotensin receptor II by clathrin-mediated endocytosis in
a temperature-dependent manner.(Pastuszka, Okamoto et al. 2014) When this ELP was fused to
caveolin, coacervation of the fusion promoted internalization of cholera toxin B in a temperature-
dependent manner.(Tyrpak, Wang et al. 2020) In addition to endocytosis, ELPs have been fused
to the epidermal growth factor receptor (EGFR) and showed temperature-dependent activation and
downstream signaling of the mitogen-activated protein kinase pathway (MAPK).(Li, Tyrpak et al.
2018) These findings have aided in understanding how microdomain formation of these specific
fusion proteins affects their biological functions. While these proteins have been extensively
studied in detail, other enigmatic proteins with vital biological functions still remained unexplored.
Flotillin-1 and -2, also known as reggie-2 and -1 respectively, are a family of ubiquitously
expressed, evolutionary well-conserved membrane raft-associated proteins sharing approximately
a 50% homology.(Babuke and Tikkanen 2007) Their multifaceted biological roles in different cell
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types have been reported extensively in literature by Nichols and colleagues;(Glebov, Bright et al.
2006, Riento, Frick et al. 2009, Ludwig, Otto et al. 2010, Otto and Nichols 2011, Riento, Zhang et
al. 2018) however, the molecular mechanisms that underlie some of their functions remain unclear.
Flotillins form homo and hetero-oligomeric complexes at the plasma membrane and are implicated
in a myriad of physiological processes such as cell adhesion, actin dynamics, endocytosis,
trafficking, signal transduction and more.(Glebov, Bright et al. 2006, Frick, Bright et al. 2007,
Babuke, Ruonala et al. 2009, Rossy, Schlicht et al. 2009, Amaddii, Meister et al. 2012, Bodin,
Planchon et al. 2014, Fork, Hitzel et al. 2014) The understanding of flotillins and their biology
made great strides since their discovery; however, human pathologies found to be correlated with
flotillin association or dysregulation such as various cancers, Alzheimer’s, diabetes or Parkinson’s
remains to be fully elucidated.(James, Cairns et al. 2001, John, Meister et al. 2014, Li, Yang et al.
2016, Chen, Wu et al. 2018, Angelopoulou, Paudel et al. 2020) Interestingly, while these disorders
seems critically dependent on flotillins, knockout mice were shown to be viable and do not have
overtly different phenotypes. (Ludwig, Otto et al. 2010, Banning, Ockenga et al. 2012, Bitsikas,
Riento et al. 2014)
Flotillin-mediated endocytosis requires the composition of both flotillin-1 and flotillin-2
complexes. Co-assembly form microdomains and can induce internalization independent of
clathrin- and caveolin-mediated endocytosis.(Glebov, Bright et al. 2006, Frick, Bright et al. 2007)
Additionally, this hetero-oligomerization is necessary to internalize cargo such as
glycophosphatidylinositol (GPI)-anchor protein CD59, and ganglioside GM1 into the intracellular
compartments.(Glebov, Bright et al. 2006, Babuke, Ruonala et al. 2009) Flotillins are typically
present as highly stable hetero-tetramers at the cell surface; however, they can be found in their
monomer forms as well.(Solis, Hoegg et al. 2007) For example, cell signaling events at the plasma
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membrane such as recruiting E-cadherin and P120-catenin to cholesterol-rich membrane domains
are regulated by flotillin-1.(Chartier, Laine et al. 2011) Also, flotillin-1 is abundantly expressed in
pyramidal neurons were found to contain mutant amyloid precursor proteins isolated from the
endosomes of postmortem Alzheimer’s patients.(Angelopoulou, Paudel et al. 2020) Thus far,
flotillins have demonstrated to have cell-type specific functions for endocytosis, trafficking or
signaling, and arriving to a conclusive explanation of their ultimate role is still an open area of
discussion.
We adopt the same technology and fuse flotillin-1 to our ELP library to determine if we
can achieve the same control as our previously published clathrin, caveolin and EGFR fusion
proteins. We demonstrate here that flotillin-1 ELP fusions are temperature-dependent, characterize
their behavior, and track their movement within the cell.

5.3. Materials and methods
5.3.1. Construction of FLOT1 plasmids
A pCMV6 plasmid encoding human flotillin-1 (NM_005803) was purchased from OriGene
(RC200231, Rockville, MD), digested with EcoRV-HF (R3195S, New England Biolabs Inc.,
Ipswich, MA) and a palindromic oligonucleotide (5’- CTGTGCAGATATCTGCACAG -3’) was
inserted. The oligonucleotide inserted contained an EcoRV blunt-end restriction enzyme
recognition site shifted forward by a single nucleotide for the in-frame insertion of the ELP genes.
pET25 vectors encoding ELP genes (V72, V96 and A96) were previously generated from the
laboratory and were excised by using NdeI and BamHI-HF (R0111S and R3136S respectively)
and blunt-ends were created with Klenow fragment (M0210S). (Shah, Hsueh et al. 2012) The
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flotillin-1 oligonucleotide inserted vector was digested with EcoRV-HF, treated with Antarctic
phosphatase (M0289S), and the ELP gene ligated with T4 DNA ligase (M0202S). Successful
insertion of the ELPs were verified by a diagnostic restriction digest using KpnI and PmeI (R3142S
and R0560S). The EGFP gene was cloned into the FLOT1-ELP plasmids by PCR of another
plasmid previously generated by the laboratory with primers containing XhoI recognition sites
(forward primer: 5’- CATCTCGAGATGGTGAGCAAGGGCGAG -3’, reverse primer: 5’-
CATCTCGAGCTTGTACAGCTCGTCCATGCC -3’, Genewiz, South Plainfield, NJ).(Li,
Tyrpak et al. 2018) The N and C terminus of the fusion gene and the linkers were verified and in-
frame by Sanger sequencing (Genewiz, South Plainfield, NJ).

Table 7. Amino acid sequence of FLOT1 constructs
FLOT1-EGFP
*MFFTCGPNEAMVVSGFCRSPPVMVAGGRVFVLPCIQQIQRISLNTLTLNVKSEK
VYTRHGVPISVTGIAQVKIQGQNKEMLAAACQMFLGKTEAEIAHIALETLEGHQ
RAIMAHMTVEEIYKDRQKFSEQVFKVASSDLVNMGISVVSYTLKDIHDDQDYLHS
LGKARTAQVQKDARIGEAEAKRDAGIREAKAKQEKVSAQYLSEIEMAKAQRDY
ELKKAAYDIEVNTRRAQADLAYQLQVAKTKQQIEEQRVQVQVVERAQQVAVQE
QEIARREKELEARVRKPAEAERYKLERLAEAEKSQLIMQAEAEAASVRMRGEAE
AFAIGARARAEAEQMAKKAEAFQLYQEAAQLDMLLEKLPQVAEEISGPLTSANK
ITLVSSGSGTMGAAKVTGEVLDILTRLPESVERLTGVSISQVNHKPLRTATRTRPL
EMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP
WPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEV
KFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHN
IEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAA
GITLGMDELYKLEQKLISEEDLAANDILDYKDDDDKV
FLOT1-EGFP-V72
*MFFTCGPNEAMVVSGFCRSPPVMVAGGRVFVLPCIQQIQRISLNTLTLNVKSEK
VYTRHGVPISVTGIAQVKIQGQNKEMLAAACQMFLGKTEAEIAHIALETLEGHQ
RAIMAHMTVEEIYKDRQKFSEQVFKVASSDLVNMGISVVSYTLKDIHDDQDYLHS
LGKARTAQVQKDARIGEAEAKRDAGIREAKAKQEKVSAQYLSEIEMAKAQRDY
ELKKAAYDIEVNTRRAQADLAYQLQVAKTKQQIEEQRVQVQVVERAQQVAVQE
QEIARREKELEARVRKPAEAERYKLERLAEAEKSQLIMQAEAEAASVRMRGEAE
AFAIGARARAEAEQMAKKAEAFQLYQEAAQLDMLLEKLPQVAEEISGPLTSANK
ITLVSSGSGTMGAAKVTGEVLDILTRLPESVERLTGVSISQVNHKPLRTATRTRPL
EMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP
96

WPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEV
KFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHN
IEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAA
GITLGMDELYKLEQKLISEEDLAANDSVQIMGVPGVGVPGVGVPGVGVPGVGVPGVG
VPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVG
VPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVG
VPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVG
VPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVG
VPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVG
VPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVG
VPGVGY
FLOT1-EGFP-V96
*MFFTCGPNEAMVVSGFCRSPPVMVAGGRVFVLPCIQQIQRISLNTLTLNVKSEK
VYTRHGVPISVTGIAQVKIQGQNKEMLAAACQMFLGKTEAEIAHIALETLEGHQ
RAIMAHMTVEEIYKDRQKFSEQVFKVASSDLVNMGISVVSYTLKDIHDDQDYLHS
LGKARTAQVQKDARIGEAEAKRDAGIREAKAKQEKVSAQYLSEIEMAKAQRDY
ELKKAAYDIEVNTRRAQADLAYQLQVAKTKQQIEEQRVQVQVVERAQQVAVQE
QEIARREKELEARVRKPAEAERYKLERLAEAEKSQLIMQAEAEAASVRMRGEAE
AFAIGARARAEAEQMAKKAEAFQLYQEAAQLDMLLEKLPQVAEEISGPLTSANK
ITLVSSGSGTMGAAKVTGEVLDILTRLPESVERLTGVSISQVNHKPLRTATRTRPL
EMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP
WPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEV
KFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHN
IEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAA
GITLGMDELYKLEQKLISEEDLAANDSVQIMGVPGVGVPGVGVPGVGVPGVGVPGVG
VPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVG
VPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVG
VPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVG
VPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVG
VPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVG
VPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVG
VPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVG
VPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVG
VPGVGVPGVGVPGVGY
FLOT1-EGFP-A96
*MFFTCGPNEAMVVSGFCRSPPVMVAGGRVFVLPCIQQIQRISLNTLTLNVKSEK
VYTRHGVPISVTGIAQVKIQGQNKEMLAAACQMFLGKTEAEIAHIALETLEGHQ
RAIMAHMTVEEIYKDRQKFSEQVFKVASSDLVNMGISVVSYTLKDIHDDQDYLHS
LGKARTAQVQKDARIGEAEAKRDAGIREAKAKQEKVSAQYLSEIEMAKAQRDY
ELKKAAYDIEVNTRRAQADLAYQLQVAKTKQQIEEQRVQVQVVERAQQVAVQE
QEIARREKELEARVRKPAEAERYKLERLAEAEKSQLIMQAEAEAASVRMRGEAE
AFAIGARARAEAEQMAKKAEAFQLYQEAAQLDMLLEKLPQVAEEISGPLTSANK
ITLVSSGSGTMGAAKVTGEVLDILTRLPESVERLTGVSISQVNHKPLRTATRTRPL
EMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP
WPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEV
KFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHN
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IEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAA
GITLGMDELYKLEQKLISEEDLAANDSVQIMGVPGAGVPGAGVPGAGVPGAGVPGAG
VPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAG
VPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAG
VPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAG
VPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAG
VPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAG
VPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAG
VPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAG
VPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAG
VPGAGVPGAGVPGAGY
* Bold indicates human flotillin-1 sequence (NM_005803). The EGFP sequence is shown in
green. Myc tag is in blue, FLAG tag is in red and ELP sequence in purple.

5.3.2. Cell Culture
HEK-293 cells (CRL-1573, ATCC, Manassas, VA) were maintained in DMEM (11965092,
ThermoFisher Scientific, Waltham, MA) supplemented with 10% FBS (35-011-CV, Corning,
Corning, NY) at 37 °C in 5% CO2 humidified tissue culture incubators. Stably transfected HEK-
293 cells were maintained with an additional 800 µg/ml of G418 selection media. At least one day
prior to conducting the experiments, the cells were placed at 31 °C in a 5% CO2 humidified tissue
culture incubator.
5.3.3. Stable cell line generation
HEK-293 cells were stably transfected with the FLOT1-containing plasmids (Table 8) by
electroporation and cell selection. Briefly, cells were grown in a T-75 flask until confluency,
trypsinized, and resuspended in 1 ml of Opti-MEM (11058021, ThermoFisher Scientific, Waltham,
MA). Then, 600 µl was transferred into a 4 mm gap sterile cuvette (45-0126, BTX, Hamden,
Connecticut) with approximately 10 ug of purified plasmid DNA in ultra-pure distilled H2O
(10977-015, Invitrogen, Carlsbad, CA) and electroporated with the BTX electro cell manipulator
(ECM 600, Hamden, Connecticut). The electroporation settings are as follows: the voltage was set
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to 500 v, the capacitance was set to 25 µF and the resistance was set to 48 ohms. Cells were
immediately seeded back into a 10 cm cell culture dish with fresh DMEM containing 10% FBS
and after 48 h, the media was replaced with DMEM containing 10% FBS with 600 µg/ml of G418
(ant-gn-5, InvivoGen, San Diego, CA) daily for 1 week. Subsequently, the media was replaced
800 µg/ml of G418 containing DMEM daily for an additional 5 days. The cells were then seeded
at 0.5 cells/well in a 96-well plate using DMEM containing 10% FBS and 800 µg/ml of G418 and
allowed to incubate at 37 °C for 2 additional weeks for monoclonal cell selection. Wells exhibiting
a change in media color (indicating cell growth) from pink to orange were observed under a
fluorescence microscope (Diaphot 300, Nikon, Melville, NY) and cells demonstrating a uniform
expression of GFP were selected for expansion, upscaling from a 6-well plate to a T-75 flask.
Stocks were prepared and stored in DMEM supplemented with 10% FBS and 7% DMSO in liquid
nitrogen.

Table 8. Summary of the fusion plasmids constructed.
Label Amino acid sequence
a

Molecular
weight
b
(kDa)
Transition
temperature
c

( °C)
Behavior
F1 FLOT1-EGFP 78 NA Normal
F1-V72 FLOT1-EGFP-G(VPGVG) 72Y 106.9 38 Temperature-responsive
F1-V96 FLOT1-EGFP-G(VPGVG) 96Y 116.8 25 < Constitutively on
F1-A96 FLOT1-EGFP-G(VPGAG) 96Y 114.1 42 < Temperature-insensitive
a
Complete FLOT1-EGFP amino acid sequence is found in table 7.

b
Expected MW is based on the ORF for the fusion construct and was calculated by ExPASy pI/Mw tool.

c
Observed transition temperature defined as no strong plasma membrane outline.

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5.3.4. Immunoblot analysis.
The stably transfected HEK-293 cells were seeded into 6-well dishes and allowed to grow until
confluency. Cells were briefly washed with ice-cold PBS and lysed with ice-cold RIPA buffer
containing a protease cocktail inhibitor (78425, Thermo Fisher Scientific, Waltham, MA) and the
samples were stored at -20 °C until use. Approximately 20 µg of total protein was loaded into a
4–20 % tris-glycine gradient gel (456-1095, BioRad, Hercules, CA) and ran at 100 v until the dye
front ran out. The protein was transferred overnight at 25 mAh onto a PVDF membrane
(ISEQ00010, Millipore, Billerica, MA) and blotted against mouse anti-myc at 1:1000 (9B11, Cell
Signaling Technology (CST), Danvers MA) in 5% BSA overnight at 4 °C. Conjugated-HRP
secondary antibody against mouse (7076, CST, Danvers, MA) at 1:5000 in 5% BSA were
incubated for 1 h at RT and the bands visualized using the SuperSignal West Pico
chemiluminescence substrate (34580, Thermo Fisher Scientific, Waltham, MA) on an iBright
FL1000 imaging system (Thermo Fisher Scientific, Waltham, MA).
5.3.5. Immunofluorescence and confocal microscopy
The stably transfected HEK-293 cell images were acquired by confocal laser scanning microscope
(LSM880, Carl Zeiss AG, Jena, Germany) with Airyscan super-resolution (63x objective lens with
a 1.4 aperture, plan-apochromatic, infinity color corrected lens). When tracking the live cell
movement, fast mode with Airyscan was selected in the Zen software to increase the scanning
speed.
5.3.6. Determination of the transition temperature by live cell imaging.
Approximately 5x10
5
of the stably transfected HEK-293 cells were seeded onto poly-D-lysine
coated 35mm glass bottom slides (P35G-1.5-14-C, MatTek Corporation, Ashland, MA) at least
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one day prior to examining the cells under the microscope. Before examining the cells under the
microscope, the cells were taken out of the incubator and allowed to sit at room RT for 1 hr. After,
the media was replaced with Live Cell Imaging Solution containing HEPES buffer (A14291DJ,
Thermo Fisher Scientific, Waltham, MA) and NucBlue Live Cell Stain ReadyProbes reagent
(R37605, Thermo Fisher Scientific, Waltham, MA) was added according to the manufacturer’s
protocol. To determine the Tt, the cells were incubated at various increasing temperatures and the
number of cells assessed under the confocal microscope. Beginning from ambient RT, 100 cells
were randomly chosen and placed under two categories: transitioned or not transitioned. The
criteria for being transitioned, is complete loss of the strong plasma membrane outline staining.
The temperature was raised within the Zeiss black software control panel and held for 20 minutes
before counting the cells.
5.3.7. Tracking internalization and colocalization
Cholera toxin B (CTxB) internalization was tracked by live cell imaging. Briefly, the stably
transfected HEK-293 cells over expressing F1-V96 was grown on a 35mm glass bottom slides as
previously described and on the day of the experiment, treated with 2 µl of CTxB conjugated to
AlexaFluor 647 (C34778, Thermo Fisher Scientific, Waltham, MA) in Live Cell Imaging Solution.
The cells were immediately tracked using the confocal microscope at low temperatures (starting
with ambient) and quickly ramped up to high temperatures. Images were automatically acquired
every 1.2 sec.

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5.3.8. Statistics
The determination of the Tt among F1 and F1-V72 were replicated 3 times on different days and
the data is presented as the absolute measured values with the dotted black lines representing the
95% confidence intervals.

5.4. Results
5.4.1. Generation of a small library of FLOT1-ELPs
V72, V96 and A96-ELPs were fused to the C-terminus of FLOT1 by traditional molecular cloning
using blunt-end ligation. Successful insertion of the ELPs into the FLOT1 backbone was verified
by a diagnostic restriction digest followed by agarose gel electrophoresis. The expected band sizes
for FLOT1, FLOT1-V72, FLOT1-V96 and FLOT1-A96 are 1401, 2533, 2893 and 2899 bp
respectively which agree with the data (Figure 29C). Successful insertion of EGFP was conducted
by PCR and the linkers were verified by Sanger sequencing. The resulting fusion proteins consist
of FLOT1, a MYC tag, the EGFP domain and the corresponding ELP (Figure 29B).
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Figure 29: Visual illustration and confirmation of FLOT1-ELPs. A) Schematic representation of the
library of flotillin-ELPs. F1 representing the wild type has normal cell behavior where movement between
the membrane and cytoplasm is rapid. F1-A96 is the temperature insensitive clone that mimics the F1. F1-
V72 behaves similarly to wild type and F1-A96 below its transition temperature but ‘switches’ to the on
position and forms large stable microdomains similar to always ‘on’ F1-V96 which does not have a strong
plasma membrane staining pattern. B) Gene’s encoding for EGFP and ELP sequences (V72, V96 and A96)
were cloned to flotillin containing MYC tag (FLAG was deleted due to ELP cloning strategy). C)
Diagnostic digest of plasmids containing FLOT1 and the ELPs of the appropriate sizes. D) Immunoblot
confirming expression and appropriate molecular weight of the stably transfected flotillins.

5.4.2. Successful generation of stable cell lines.
HEK-293 cells were stably transfected by electroporation and antibiotic selective pressure
maintained FLOT1-ELP expression over several months. The appropriate molecular weight and
expression of each flotillin construct was verified by immunoblot analysis (Figure 29D).
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5.4.3. Determination of the transition temperature.
Each of the F1-ELPs were observed using live cell microscopy and movements tracked by the
ocular lens. Overexpression of F1 exhibits a dispersed staining pattern at the plasma membrane
creating a strong outline of the cell. F1-V72 has the same staining pattern at physiological
temperatures as well as temperature insensitive F1-A96. When increased above 37 C, over a
period, F1-V72 lost its outline and the microdomains tended to travel slower, whereas F1 and F1-
A96 still had the dispersed staining to outline the plasma membrane. The cells expressing F1-V96
population did not have any plasma membrane outline. Moreover, the puncta looked to be stuck
on ‘tracks’ and did not move rapidly as wild type, F1-A96 or F1-V72 below physiologically
relevant temperatures. Cells were counted as ‘transitioned’ or ‘non-transitioned’ under the ocular
lens of the confocal microscope and the total percent was extrapolated onto a graph (Figure 30A).
Based on these observations, F1-A96 was classified as temperature-insensitive, behaving
identically to wild type. F1-V96 on the other hand, was classified as constitutively ‘on’, meaning
the ELPs phase separated at all the temperatures measured down to 25 C. F1-V72 was classified
as temperature responsive because at temperatures below the predicted Tt, F1-V72’s phenotype
matched that of F1. Above 37 C, the phenotype tended more to match F1-V96 phenotype. All of
the phenotypes at low and high temperatures can be found in Figure 30B. F1-V72’s percent
transitioned was plotted over temperature and a nonlinear exponential growth equation was used
to match the data. Afterwards, a linear regression was plotted and extrapolated corresponding to
the maximum first derivative. The line that crossed F1 as the negative control was considered to
be the Tt of F1-V72, which was 38 C.
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Figure 30: Cell staining pattern of FLOT1-ELPs represents different behaviors. A) A graph showing
the percent of each flotillin construct transitioned at different temperatures. B) Representative images of
the plasma membrane staining pattern at low or high temperatures. Wild-type F1 at low or high
temperatures maintains a solid outline of the plasma membrane. F1-V72 at low temperatures has a solid
outline. Increasing the temperature above 38 °C loses the solid outline at the plasma membrane. F1-V96 at
low or high temperatures never has a solid outline of the plasma membrane. F1-A96 at low or high
temperatures always maintains a sold outline. Refer to table 8 for their behavior.

5.4.4. Cholera toxin B colocalizes with F1-V96 and is sent to the lysosome.
Immediately after the addition of CTxB, F1-V96 and CTxB was tracked by live cell microscopy.
In the beginning at room temperature (25.8 C), the HEK-293 cells can be seen rapidly
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internalizing CTxB (red) from the plasma membrane, especially at areas with high levels of F1-
V96 (green) (Figure 31A). Colocalized internalization is seen at the ends of the cell body in yellow.
Over a period of 15 min, the temp is increased where the temp of the media is measured as 39 C.
Most of the co-localization is now within the cell found in a large organelle-like structure. We
identified these large organelles in the cytoplasm by immunostaining internalization markers and
showed that F1’s associate in these structures containing LAMP1 which is a marker for the
lysosome (Figure 31B).

Figure 31: Cholera toxin subunit B is internalized and colocalizes with F1-V96. A) CTxB (red)
colocalizes with F1-V96 (green) at the plasma membrane (25.8 C) and both shuttle to the lysosome over
a short period during a temperature ramp (39 C). B) LAMP1 (red), a lysosomal marker, colocalizes with
overexpressed F1-ELPs (green) having a large organelle appearance in the cytoplasm. Nuclei stained in
blue.
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5.5. Discussion
The flotillin family consisting of flotillin-1 and flotillin-2 are a multifunctional, raft-associated
scaffolding proteins involved in a variety of biological processes such as flotillin-mediated
endocytosis, trafficking, signal transduction, actin mobility and more (Kwiatkowska, Matveichuk
et al. 2020). Their protein expression level varies based on cell type and have different functional
roles (Langhorst, Reuter et al. 2005). Within the past decade, a plethora of evidence have been
uncovered demonstrating a correlation with flotillin dysregulation and disease (Bodin, Planchon
et al. 2014); however, double knockout mice show no phenotypic abnormality (Otto and Nichols
2011). This may lead many to believe that there are compensatory mechanisms that can take place
of flotillins. Such uncertainty warrants new tools to attempt to delineate this convoluted
understanding of flotillins.
Modern advances in protein engineering have enabled us to utilize the potential of specific
domains and fuse them to generate a fusion with multi-functional properties. Here, we created a
small library of flotillin-ELPs, each having a different characteristic. F1-V72 is temperature
responsive, F1-V96 is constitutively ‘on’, while F1-A96 is the temperature insensitive version.
The rationale for developing multiple constructs is to have temperature controls (A96) and to ‘tune’
the transition temperature. Overexpression and oversaturation of these F1-ELP fusion
microdomains behave similarly to previous research of the overexpressed wild-type flotillins
suggesting that the ELP tail does not interfere with shuttling between the plasma membrane or
cytoplasmic space (Frick, Bright et al. 2007, Babuke, Ruonala et al. 2009). The same ELP moiety
may work well with one protein, but when fused to another protein, may behave completely
different due to the nature of these biopolymers. F1-V72 has an estimated Tt of 38 C which is
useful for growing cells in culture; however, prolonged hyperthermia induces metabolic changes
107

to the cell and increases the risk of cell death. F1-V96 had a punctate pattern plasma membrane-
less outline even at 25 C and has largely has a different phenotype compared to the others. When
determining the Tt, observations through the ocular lens or eye piece of the microscope suggested
that microdomain formation or phase separation of F1-V72 requires more time than other ELP-
fusions we have worked with in the past. One possible explanation for this is that all these fusions
are shuttling around from the plasma membrane back to the cytoplasm anchored to lipid bilayer.
Therefore, the ELP tails may not immediately be available to phase separate until enough come
together. This also might explain why the strong outline disappears when incubated above the Tt.
Below the Tt, the lipid rafts freely flow through the plasma membrane like a loose buoy in the sea;
however, when above the Tt, while they shuttle back into the cytoplasm, more join with the larger
condensate also corroborating our rationale.
Interestingly, when CTxB is introduced to F1-V96 even at above its Tt, there is still
colocalization at the plasma membrane and they internalize together. This suggests that F1-V96,
while constitutively ‘on’, can still shuttle between the plasma membrane and the inside of the cell
and additionally, that the F1-ELPs can interact with endogenous F2 to promote internalization of
GM1. Moreover, they shuttle together and are sent to the lysosome. While these fusions enable
live tracking and control of F1’s through their ELP domain, further characterization is required to
completely understand the fusion proteins’ phase behavior. Without a complete understanding of
the fusion protein, interpretation of the results may be equivocal. This preliminary result
demonstrates the usefulness of this technology and may be used to further interrogate F1
trafficking and cell signaling.

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5.6. Summary
For this chapter, a library of ELPS (V72, V96 and A96) were genetically fused to flotillin-1 and
stable cell lines were generated. Each construct had a different phenotype: thermally responsive,
constitutively ‘on’ and temperature insensitive. F1-V96 internalized and shuttled CTxB to the
lysosome via GM1.
CHAPTER 6: SUMMARY, CHALLENGES AND FUTURE PERSPECTIVES
6.1. Summary
Cell communication is facilitated by an orchestra of biomolecules found in and outside of the cell
and is comprised of an extensive network that self-governs all the biological activities.
Understanding these intricate pathways enable us to answer important questions about the
pathophysiological pathways involved in many fatal diseases among humans. Thus, intense
biomedical research has focused on discovering novel methods utilized to support, characterize
and fully comprehend these routes.
In this dissertation, we examine three different major pathways: laminin, estrogen receptor
and flotillin. To study these groups, we use ELP repertoire as the domain which are stimuli-
responsive and drive phase separation and microdomain formation.
Specific laminin sequences have been identified as having biological activity; however,
their heterotrimeric structure is too large to purify and their minimal recognition sequence is too
small to adhere. Therefore, we used I30-ELP as the backbone to promote laminin signaling of
fibroblast skin cells. These peptides, when coupled to ELPs provide the necessary signals for cell
adhesion and spreading.
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Estrogen receptor has been extensively studied as a transcription factor and regulates many
processes; however, there are many rapid responses that cannot be explained by regulating
transcription alone. Consequently, research focusing on ERα rapid signaling has gained popularity
but traditional methods using small molecule inhibitors have does not have the specificity to
control cytoplasmic signaling. When ELPs are fused to ERα they form microdomains when
stimulated above the Tt and can act as a switch.
Flotillins are ubiquitously expressed proteins that have been associated with and used as
prognostic markers for various pathological diseases. While plenty of research has described its
biological function such as endocytosis, trafficking and cell signaling, we are still viewing flotillins
at the tip of the iceberg. Flotillins are multifaceted and do not have direct small molecule inhibitors,
so knockdowns are primary choice; however, they cannot be rapidly reversed. When flotillins are
fused to ELPs, present as 3 major phenotypes and may be used to modulate the movement within
cells.
Synthetic ELP biopolymers introduced in this dissertation has been the cornerstone for
these projects because their biophysical properties enable self-assembly and phase separation.
They are a genetically encodable and tunable platform that which allows for full customization
can be appended to any domain or protein adding dual function to the hybrid or fusion. Their phase
separation is rapid and reversible and they have low immunogenicity. Overall, their unique
characteristics make them an excellent choice for materials and as molecular switches.
6.2. Challenges
While ELPs have favorable characteristics, there are some concerns that need to be
addressed. First of all, while ELPs can be purified in large quantities in bacteria, E. coli does not
110

typically contain the necessary post-translational modifications that mammalian proteins are
subjected to. Furthermore, their purification process using ELP-mediated phase transition, require
a lot of manual labor and would benefit from having the purification process automated. Next,
when making ELP fusions, while most behave in a similar manner, all fusions must go through a
characterization step because sequence that is attached to the ELP may promote the formation of
different structures when induced to phase separate. Another issue with ELPs are because of the
stimuli required itself- heating and cooling cells to induce microdomain formation adds another
layer of controls that need to be taken into consideration. Furthermore, it is difficult to apply ELP
as molecular switches in a translational manner since only the surface of the body can be heated
or cooled down. While these pose as significant challenges to generate fusion ELPs with
multifunctional properties, careful planning and the right application can overcome these
challenges.
6.3. Future perspectives
ELPs are hydrophobic domains, inspired from human tropoelastin from the extracellular matrix
and have exceeded the expectation for use as a biomaterial for cell culture and tissue engineering.
A hydrogel using ELPs, collagen-like polypeptides and a variety of extracellular signals all in one
place can be the perfect recipe for a biosynthetic membrane in tissue regeneration and wound
healing.

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Abstract (if available)

Abstract This dissertation harnesses the power of synthetic biology and protein engineering to generate peptide and protein fusions that strategically employs stimuli-responsive biopolymers called elastin-like polypeptides (ELPs). ELPs are a class of biopolymers that undergo a reversible phase separation in response to heat where they self-assemble to form an insoluble coacervate. This unique characteristic, when coupled to a domain or protein can drive biomolecules to concentrate and can modify the properties or behavior of fusion. The bioactivity of these complexes are explored in this dissertation where these fusions with multi-functional properties are characterized. In Chapter 1, a proof-of-concept using a short hydrophobic ELP (I30) with a biologically active laminin peptide (A99) from the extracellular matrix is produced. The fusion is compared against various conditions and demonstrate to have favorable biophysical properties for purification and bioactivity in cell adhesion and spreading. Chapter 2 expands on this technology and introduces a library of laminin peptides (A2G80, AG73 and EF1m) from different domains of laminin fused to the ELP. Their biological activity in the context of cell attachment and spreading is assessed and shown to have synergy when mixed. Additionally, they promote neurite outgrowth of neuronal cells. Chapter 3 moves into the cell and studies how classic nuclear hormone receptor, estrogen receptor α (ERα) behaves when fused to an ELP. At temperatures below 23℃, ELP-ERα fusions remain soluble both in the cytoplasm and the nucleus; however, with the addition of estradiol promotes concentration and microdomain formation which is found predominantly in the cytoplasm. Finally, in Chapter 4, a less understood multi-functional protein called flotillin-1 is fused to a library of ELPs and are briefly characterized. Flotillin-ELP fusions mobilize rapidly throughout the cell and shuttle between the cytoplasm and plasma membrane. These chapters demonstrate how ELPs unique characteristics may modulate biologically relevant pathways.

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Asset Metadata

Creator Truong, Anh Tan (author)

Core Title Synthetic biopolymers modulate cell signaling

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Molecular Pharmacology and Toxicology

Degree Conferral Date 2021-12

Publication Date 10/11/2021

Defense Date 05/13/2021

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag elastin-like polypeptide,estrogen receptor,flotillin,laminin,OAI-PMH Harvest,synthetic biology

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor MacKay, Andrew (committee chair), Okamoto, Curtis Toshio (committee chair), Alashkar, Houda (committee member), Hamm-Alvarez, Sarah (committee member), Stiles, Bangyan (committee member)

Creator Email intekmdma@gmail.com,truonga@alumni.usc.edu

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