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Physical chemistry and polymeric modification of elastin-like polypeptides

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Physical chemistry and polymeric modification of elastin-like polypeptides

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PHYSICAL CHEMISTRY AND POLYMERIC MODIFICATION OF
ELASTIN-LIKE POLYPEPTIDES
By
Jiawei Wang

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)
May 2014

Copyright 2014 Jiawei Wang

II

ACKNOWLEDGEMENTS
Foremost, I would like to express my sincere gratitude to my advisor Dr. Andrew
MacKay for providing me the opportunity of such an innovative research project. I would
like to thank him for all the continuous support, his guidance and encouragement as well
as his patience and broad scope of knowledge. I would also like to thank Dr. Cesar
Blanco and Dr. Mark Thompson for their precious advices and support on this project.
My sincere thanks also go to Dr. Manuel Orosco for all his help in designing experiments
with specialized organic chemistry knowledge and training me on the necessary
techniques.
Additionally, I would like to thank my colleagues in Dr. Andrew MacKay’s lab, Dr.
Suhaas Aluri, Dr. Siti Mohd Janid, Wan Wang, Pu Shi, Martha Pastuszka, Jugal
Dhandhukia, Zhe Li, Yuqian Liu, Jordan Despanie and Dab Brill for all the great
cooperation and their generous support. In particular, I’m grateful to Dr. Joshua
Gustafson for his valuable advices not only for experiments but also for my scientific
career.
I would also to thank my committee members Dr. Bogdan Olenyuk and Dr. Curtis
Okamoto for their brilliant comments and suggestions as well as the precious time they
spent on reviewing my thesis. Moreover, I would like to thank the USC school of
Pharmacy as well as Alfred-Mann Institute for supporting to this project.

III

Last but not least, a special thanks to my family. Thanks for all the sacrifice my mother
and father have made on my behalf. I would also like to thank all my friends, Yiyang
Yuan, Li Zhou, Ning Ning, Xingliang Zhou, Chenchen Yang, Yanke Lu, etc. for their
spirit support during my two-year master’s study in Los Angeles.

IV

TABLE OF CONTENTS

ACKNOWLEDGEMENTS .......................................................................................... II
LIST OF FIGURES ..................................................................................................... VI
LIST OF TABLES ..................................................................................................... VII
ABBREVIATIONS ................................................................................................... VIII
ABSTRACT .................................................................................................................. X
CHAPTER 1 .................................................................................................................. 1
INTRODUCTION AND BACKGROUND ............................................................... 1
1.1 Elastin-Like Polypeptides (ELPs) ..................................................................... 1
1.2 Characterization of Polymers: Thermal Analysis ............................................. 3
1.3 Fragile Skin and Ideal Bandage Materials ........................................................ 3
1.4 Pressure Sensitive Adhesives (PSAs) ............................................................... 4
1.5 Polymerization Mechanism .............................................................................. 6
1.6 Polymerization Techniques ............................................................................... 8
CHAPTER 2 ................................................................................................................ 12
MA TERIALS AND METHODS .............................................................................. 12
2.1 Material ........................................................................................................... 12
2.2 ELP expression and purification using ITC .................................................... 12
2.3 ELPs Transition Temperature and Concentration Determination by UV-Vis
Spectrophotometer ................................................................................................ 14

V

2.4 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) 14
2.5 Differential Scanning Calorimetry (DSC) ...................................................... 15
2.6 Polymerization of ELP .................................................................................... 15
CHAPTER 3 ................................................................................................................ 17
RESULTS ................................................................................................................. 17
3.1 Confirmation of purity and correct molecular weight of selected ELPs by
SDS-PAGE ............................................................................................................ 17
3.2 Determination of Concentration and Tt of ELPs by UV-Spectrophotometer . 18
3.3 Thermal analysis of ELPs by Differential Scanning Calorimetry .................. 20
3.4 PSA-ELP conjugation confirmation by FTIR-ATR ........................................ 27
3.5 Compare Tt of polymerized ELPs by UV-Spectrophotometer ....................... 29
3.6 Determination of accurate molecular weight of ELPs by MALDI ................. 30
CHAPTER 4 ................................................................................................................ 32
DISCUSSION .......................................................................................................... 32
CHAPTER 5 ................................................................................................................ 35
FUTURE DIRECTIONS .......................................................................................... 35
REFERENCES ............................................................................................................ 36

VI

LIST OF FIGURES
Fig.1: The ELP phase transition. .................................................................................... 2
Fig.2: Generic demonstration of step-growth polymerization(Cowie, 2008) ................ 7
Fig.3: Reaction mechanism of acrylate based chain polymerization (G, 2004) ............ 7
Fig.4: Schematic representation of emulsion polymerization........................................ 9
Fig.5: SDS-PAGE of selected ELP. ............................................................................. 17
Fig.6: The wavelength scans of V96. ........................................................................... 18
Fig.7: The Tm analysis of V96. ................................................................................... 19
Fig.8: Compare the Tt difference between three different kinds of ELPs. .................. 20
Fig.9: Transition phase of V96 measured by DSC. ..................................................... 21
Fig.10: Compare of the difference in transition temperatures measured by two different
ways. ............................................................................................................................ 24
Fig.11: Compare the different ΔH of ELP with Isoleucine at different lengths. .......... 25
Fig.12 : Compare the different ΔH of ELP with Valine at different lengths. ............... 26
Fig.13: Compare the difference in absorbance peaks between pure V96 and V96 coupled
with initiator ................................................................................................................. 28
Fig.14: Compare the difference in absorbance peaks between V96-Initiator and
polymerized V96 .......................................................................................................... 29
Fig.15: Tm analysis of V96, V96-Initiator and polymerized V96 ............................... 30
Fig.16: MALDI measurement of ELPs. ....................................................................... 31

VII

LIST OF TABLES
Table 1: Structure of three common kinds of acrylate monomers of pressure sensitive
adhesives ........................................................................................................................ 6
Table.2: Statistic analysis of the data presented in Fig.11 ........................................... 25
Table.3: Statistic analysis of the data presented in Fig.12 ........................................... 26

VIII

ABBREVIATIONS
DCC N, N’-Dicyclohexylcarbdiimide
DIPEA Di-isopropyl Ethyl Amine
DSC Differential Scanning Calorimetry
E.Coli Escherichia Coli
EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
EHA 2-ethyl hexyl acrylate
ELPs Elastin-Like Polypeptides
FTIR-ATR Fourier Transform Infrared Spectroscopy
Attenuated Total Reflectance
ITC Inverse Transition Cycling
KDa Kilo Dalton
LCST Lower Critical Solution Temperature
MALDI Matrix Assisted Laser Desorption/Ionization
NHS N-Hydroxy Sccinamide

IX

PEI Polyethylene Terephthalate
PSA Pressure Sensitive Adhesive
RAFT Reversible Addition-Fragmentation chain
Transfer
SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel
Electrophoresis

Tt Transition Temperature
V48 G(VPGVG)
48
Y
V96 G(VPGVG)
96
Y
I24 G(VPGIG)
24
Y
I48 G(VPGIG)
48
Y
I96 G(VPGIG)
96
Y
A96 G(VPGAG)
96
Y
A192 G(VPGAG)
192
Y

X

ABSTRACT
Polymers are widely used in biomedical engineering. Among these, protein polymers like
elastin-like polypeptides (ELPs) have emerged with multiple novel applications. In this
study, we have evaluated the potential application for ELPs to develop reversible
adhesives for use with medical bandages. A current limitation of bandages is that they
continue to adhere even with the bandage must be removed, and for patients with fragile
skin bandage removal can actually create new wounds. To overcome this problem, new
bio-adhesive materials are necessary. ELP protein polymers phase separate above a
transition temperature, which can be tuned by changing their amino acid composition. In
order to understand how ELP polymers might be used to generate thermally-reversible
adhesives, my thesis has focused on investigating their physical chemistry as well as their
modification by adhesive polymers. In the first part of this study, efforts were made to
figure out the change in entropy during the transition process for various kinds of ELPs
by using differential scanning calorimetry. In the second part of the study, emulsion
polymerization method was used to polymerize ELP with acrylate based pressure
sensitive adhesives (PSAs). From current results, it seems the delta H in transition
process of ELPs is not only dependent on the type of guest residues but also on the length
of ELPs. The polymerization process has not been successed yet. In the end, future
directions are also discussed.

1

CHAPTER 1
INTRODUCTION AND BACKGROUND
1.1 Elastin-Like Polypeptides (ELPs)
Elastin-Like Polypeptides are a class of temperature-sensitive, recombinant biopolymers
derived from human tropoelastin. ELPs are composed of pentapeptide (VPGXG)n repeat
units, where X is any amino acid except proline while n represents the number of repeats
encoded.(J.Andrew MacKay, 2010) ELPs have characteristic phase transition
temperatures (Tt) below which they are soluble in water whereas above which they form
an insoluble amorphous state (as shown in Fig.1). This phase transition is completely
reversible. ELPs provide many benefits that are unique to genetically engineered
biopolymers. First of all, ELPs are non-toxic, biocompatible and biodegradable as they
consist of amino acids.(Shah et al., 2012) In addition, ELPs have favorable
pharmacokinetic profiles.(Janib et al., 2013) What’s more, the composition of ELPs can
be precisely encoded at the gene level, which means the molecular weights of ELPs can
be precisely controlled by using genetic engineering techniques.(Meyer, 2004) Last but
not least, ELPs can be easily expressed at high yield (100-200 mg/L) in E. coli and
rapidly purified by Inverse Temperature Cycles (ITCs) (Aluri et al., 2009; McDaniel et al.,
2010). Upon lowering the temperature below Tt, ELPs become less ordered which results
in relaxation of polymer. A mechanical model of energy transfer has been represented by

2

such contraction and relaxation of ELPs. Briefly, the characteristic inverse transition
temperature of ELPs governs their hydrophobic folding based on the principles of free
energy transduction.(Urry, 1997)

Fig.1: The ELP phase transition.
Fig.1: (VPGIG)
48
Y , changes from a soluble state to an insoluble state around a transition
temperature (Tt). (Image Courtesy of Dr. Siti M. Janib)
The transition temperature (Tt) of ELPs can be tuned by varying the guest residues ”X”
and number of repeats n in the (VPGXG)n sequence. (Urry, 1997) Also, concentration,
pH, and salt concentration could affect Tt of ELPs. Taking this unique property into
consideration, ELPs were selected to develop a novel kind material that has
temperature-dependent adhesiveness.

3

1.2 Characterization of Polymers: Thermal Analysis
Despite decades of research, only several papers have looked at the thermodynamics of
the temperature-mediated ELP phase separation. Operating under the assumption that an
enhanced understanding of the mechanism driving ELP phase separation. (Tirrell, 2003)
Seven ELPs (I24, I48, I96, V48, V96, A96, and A192) were characterized using
Differential Scanning Calorimetry (DSC). DSC is a widely used technique to study
thermal-induced phase transitions in concentrated materials. It provides direct,
model-independent and precise information about transition temperature, enthalpy of
transition and a type of transition (endothermic vs exothermic). Also, DSC is significantly
useful in determining the glass transition temperature (Tg), a second transition phase for
ELPs of synthetic as well as protein polymers (Gholap, 2012).
Different phase transitions of ELPs on various concentrations are effectively studied by
the measurement of DSC. When ELPs are dissolved in PBS, the ELP phase separation is
an endothermic process during heating above Tt.
1.3 Fragile Skin and Ideal Bandage Materials
It’s a growing concern throughout the world about the skin care treatments that could
benefit people with fragile skin, such as the elderly, burn victims, patients with pressure
ulcers, deep surgical wounds and nevertheless patients with rare genetic conditions.
(Gupta, 2010) One of the most useful therapeutic aids is the elastic bandage, especially

4

for the treatment for people suffered from small burns on extremities. The elastic bandage
fixes the dressing materials and provides mechanical protection to prevent irritation of the
wound by movement. (Y.Sawada, 1992) Bandages based on natural polymer are
biodegradable and give gentle touch to wounds; however, they need frequent change of
dressing because they retain foreign material and dead tissues. Compared to natural
polymer based bandages, bandages based on synthetic polymers are more stable and can
be modified structurally as well as functionally; however, most of synthetic polymers are
non-biodegradable. Additionally, both natural and synthetic polymers benefit from extra
medication to relieve pain and irritation at wounded tissue site. (Ajay V .Singh, 2010)
For traditional wound coverage materials, the key qualities include bacteriostatic,
fungistatic, non-toxic, highly absorbent, non-allergenic, breathable, hemostatic,
biocompatible, and suitably compliant mechanical properties. The occlusive dressing
provides a moist environment and maintains a homeostatic environment around injured
tissue. For example, foams, hydrofibers, crystalline sodium chloride gauze, calcium
alginates, hydrogel and hydrocolloids are traditionally used as occlusive dressings.
Recently, nanomaterial attracted much attention among clinicians and researchers
because they have new potential applications in biomedicine. (Ajay V .Singh, 2010)
1.4 Pressure Sensitive Adhesives (PSAs)
Pressure Sensitive Adhesives (PSAs) are polymers whose adhesiveness would increase

5

upon certain pressure.(Feldstein and Siegel, 2012). The most common applications of
PSAs are tapes, labels and protective films. The PSA performance is determined by three
general adhesive properties which are tack, peel strength, and shear strength. Peel
strength is the force required to remove a standard PSA strip from a specified test surface,
which is measured under a standard test angle (90°, 180°) with standardized condition.
Sheer strength is the internal or cohesive strength of the adhesive mass, which is
determined as the length of time it takes for a standard strip of PSA to fall from a test
panel after application of a load. The balance, which is usually tailored according to the
end use of the PSA, between these three major, interrelated properties result in the PSA
(Jovanović and Dubé, 2004)
Those ones based on acrylate copolymers are considered the most promising PSAs.
Acrylate pressure sensitive adhesives are the most widely used pressure sensitive
adhesives for skin care treatment. These acrylate pressure sensitive adhesives are acrylic
esters which would turn to soft and tacky polymers. Those new formed polymers possess
low glass transition temperature. By forming physical bonds such as dipole induction,
dipole-dipole forces, hydrogen bonds and Van der Waal’s forces, those acrylate pressure
sensitive adhesives adhere to skin instantaneously when applied. The physical bonds and
polymer properties are dependent on the types of monomer chosen for synthesis.
Generally, three kinds of acrylate pressure sensitive adhesives are used for skin care,
which include 2-ethyl hexyl acrylate (EHA), butyl acrylate and iso-octyl acrylate

6

(Table.1). The homopolymers of those three have glass transition temperatures (Tgs)
below 0 ℃ and inherent tackiness while lacking sufficient cohesive strength (Jovanović
and Dubé, 2004). In our experiments, 2-ethyl hexyl acrylate is chosen because it has
better polymer characteristics and easy availability.(Gholap, 2012)

Table 1: Structure of three common kinds of acrylate monomers of pressure sensitive
adhesives (Gholap, 2012)
1.5 Polymerization Mechanism
Generally, polymerization process is classified to step polymerization and chain
polymerization. Step polymerization refers to a type of polymerization mechanism in
which monomers form dimers first, then trimmers, longer oligomers and eventually long
chain polymers (as shown in Fig.2). Branched polymer might be produced as there’s the
possibility that the monomer has two reactive sites. Many natural polymers and some
synthetic polymers are produced by step-growth polymerization mechanism.(Cowie,
2008)

7

Fig.2: Generic demonstration of step-growth polymerization(Cowie, 2008)

On the other side, chain polymerization needs an initiator to start the chemical process.
The function of the initiator is to form reactive species such as free radicals. Further
polymerization is called chain propagation which is a process that a reactive intermediate
is continuously regenerated by adding a new monomer molecule present in the mixture.
So chain polymerization is also called free radical polymerization. For the acrylate
Fig.3: Reaction mechanism of acrylate based chain polymerization (G, 2004)

8

monomers we used in groups at one end, they follow free radical polymerization by
generating reactive free α-carbons which further attack β-carbons of other monomers thus
propagate the chain polymerization.(Gholap, 2012) (Fig.3)
1.6 Polymerization Techniques
Generally, acrylate polymerization techniques could be classified as solution
polymerization and emulsion polymerization. For solution polymerization, it is a free
radical polymerization in homogenous solution as its name indicates. The solution
polymerization is termed as precipitation polymerization if the polymer is insoluble in the
solvent (Gholap, 2012).
From the perspective of the overall situation, emulsion polymerization is classified as a
heterogeneous free-radical chain growth polymerization. It is a technique in which water
insoluble monomers are dispersed as small droplets. In an emulsion polymerization,
emulsifiers are widely used to stabilize small droplets of monomers and to form micelles,
in which some monomers would dissolve and almost 99% of particulate nucleation of
chain polymerization occurs. Along with the chain polymerization continues, the
monomers from the droplets would diffuse into micelles(Gholap, 2012) (Fig.4). Broadly,
both the polymerization rate and the molecular weight of polymer could be increased
simultaneously in the emulsion polymerization. It’s essential for pressure sensitive
adhesives to be fluid at the bonding and application temperature (Jovanović and Dubé,

9

2004).

Fig.4: Schematic representation of emulsion polymerization
(Courtesy Vinit Gholap)
1.6.1 DCC standard synthesis
N, N’-Dicyclohexylcarbodiimide (DCC) is an organic compound. The primary use of
DCC is to couple amino acids during artificial peptide synthesis. It exists in the form of
white crystals with a heavy, sweet odor under standards conditions. DCC has low melting
point which allows for its easy handling. DCC is a dehydrating agent for the preparation
of amides, ketones, nitriles. It would hydrates to form dicyclohexylurea (DCU), which is
insoluble in most organic solvents and in water.

10

DCC is used widely in peptide coupling. During our protein-polymer coupling, the
N-terminus is used as the attachment site on which PSA monomers are added. The
function of DCC is to enhance the electrophilicity of carboxylate group. The central
carbon in DCC would be attacked by the negatively charged oxygen which acts as a
nucleophile. As a result, DCC is temporarily forming a highly electrophilic intermediate
by attaching to the former carboxylate group. So the chain polymerization would be more
efficient on adding monomers.(PriBar, 1997)
1.6.2 ATRP and RAFT Polymerization
Atom transfer radical polymerization (ATRP) is one of living polymerization. ATRP
reactions could tolerant many functional groups like allyl, amino, epoxy, hydroxyl and
vinyl groups so they are very robust in either the monomer or the initiator. As ATRP
methods are easy prepared, commercially available and the initiators needed are
inexpensive, it is widely used in polymerization process.(Cowie, 2008)
Reversible Addition-Fragmentation chain Transfer (RAFT) is one of the controlled
radical polymerizations. RAFT polymerization uses thiocabonylthio compounds, like
thiocarbamates or xanthates to mediate the polymerization via a reversible chain-transfer
process. RAFT could still functional in aqueous solution while ATRP could not. Also,
RAFT could be effectively carried out over a wide temperature range.(David B. Thomas,
2004)

11

In this study, both ATRP and RAFT methods have been applied to the polymerization
research of ELPs.

12

CHAPTER 2
MATERIALS AND METHODS
2.1 Material
Plasmids constructs of ELPs were selected from ELP library from Dr. Andrew MacKay’s
laboratory. ELPs were obtained and purified from BLR competent cells (EMD Millipore)
cultured in TB media (Mo-Bio Laboratories, Inc). 2-Ethyl hexyl acrylate (Sigma-Aldrich)
was used as Pressure Sensitive Adhesive (PSA). 2-Chloropropionic Acid,
2-Chloropropionl Chloride, Acryloyl Chloride and
4-Cyano-4-(Dodecylculfanylthiocarbonyl) Sulfanylpentanoic Acid were used as initiators
in emulsion polymerization. Coupling agents required for protein conjugation reaction,
viz. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N,
N’-dicyclohexylcarbodiimide (DCC), N-Hyroxysccinimide (NHS) were obtained from
Thermo Scientific Inc. N, N-Diisopropylethylamine (DIPEA) was used as
non-nucleophilic base in conjugation reaction.
2.2 ELP expression and purification using ITC (Hassouneh et al., 2012)
Plasmid constructs of desired ELPs were selected from ELP library and transformed into
freshly thawed BLR competent cells. After overnight incubation in the 37 ℃ incubator on
agar plate, single bacterial colony was picked and inoculated into 50ml starter of TB
media. After overnight incubation in the 37℃ shaker, bacterial culture was centrifuged at

13

4000 rpm for 10min. The pellets were re-suspended in 5 ml fresh TB media and
inoculated into 9 liters of culture media. 0.5 ml of the re-suspended solution would be
mixed with 0.5 ml 7% DMSO for future stock preserved in -60℃ freezer. After overnight
incubation at 37℃, the culture was centrifuged at 4000 rpm for 10 min and pellets were
re-suspended in around 200 ml fresh cold PBS. These re-suspended pellets were
sonicated on ice for 15 min with the intensity level of 11.
Cold Spin: After sonication, lysed bacterial cells were centrifuged at 12000 rpm, 4℃ for
15 min in Oakridge centrifuge tubes. The supernatant was collected and the pellets were
discarded. Polyethylenimine (PEI) with a final concentration of 0.25% was added to the
collected supernatant. The mixture was placed on ice for 20 min then was centrifuged at
12000 rpm, 4℃ for 15min. Again the supernatant was collected and the pellets were
discarded.
Hot Spin: The supernatant was incubated at 37 ℃ for 10min. If necessary, 1-2M of
sodium chloride was added to the supernatant to make sure all the ELPs have been
transited to turbid status. The turbid mixture was concentrated at 4000 rpm, 37℃ for 10
min. After the hot spin, the supernatant was discarded and the pellet was re-suspended in
cold PBS.
The re-suspended pellet was again subjected to cold spin to get rid of insoluble debris.
This cycle between cold and hot spins was repeated five times to get 95% purity.

14

2.3 ELPs Transition Temperature and Concentration Determination by UV-Vis
Spectrophotometer
ELPs transition temperature (Tt) was determined over the different guest residues, the
different numbers of pentamer repeats and the different concentrations. A temperature
ramp was run from 10℃ to 80℃ at the scan rate of 1 ℃/min. Optical density was
measured at 350 nm. Tt was measured as midpoint of turbidity curve.
ELPs were run with the wavelength from 200 nm to 800 nm under the temperature of
12℃. As the absorbance of sample was determined by the equation below:
A= εlc (A is absorbance at certain wavelength; εis constant which is 1280 here; l is the
thickness of the cuvette which is 1 here; c is the concentration of samples)
So the concentration of ELPs should be calculated by:
Conc (M) = (A280-A350)/1280
2.4 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Purified proteins were run on a SDS-PAGE to confirm the molecular weight as well as
the purity. Lonza PAGEr® Gold Precast 4-20% T-G gels (Fisher Scientific Inc.) were
used this experiment. 1X SDS solution diluted from 10X SDS solution was used as
running buffer in electrophoresis chamber. 4X SDS solution containing glycerol and
bromophenol blue was used as loading sample buffer. ELPs mixture was denatured by

15

heating to 95 ℃ for 5 min followed load into wells. 5 ul Protein marker (New England
Biolabs) was loaded on both side wells of the sample. Electrophoresis was run at 120 mV
for 1 1/2 hour. Cupric chloride solution (5 g/50 ml) was used for half hour to stain the
protein bands.
2.5 Differential Scanning Calorimetry (DSC)
Thermal transitions of the different kinds of ELPs were studied using differential
scanning calorimeter (DSC 8000 Perkin Elmer Inc.). 20 ul of ELP samples were placed in
the DSC pan and 20 ul PBS was also placed in the DSC pan as blank control. All the
samples were run from 4 ℃ to 80℃ with the rate of 1 ℃/min.
2.6 Polymerization of ELP
Carboxyl groups of PSA polymeric domains were conjugated to amine groups of ELP
V96 using coupling agents N, N’-dicyclohexylcarbodiimide (DCC) and
N-Hydroxysuccinimide (NHS). This is a two-step polymerization process. The first step
is coupling ELP with the initiator. The ratio of ELP: Initiator: DCC is 1: 10:10.
Everything dissolved in anhydrous DMSO separately before mixed. The coupling
chemistry was carried at room temperature for six hours. Water was added to stop the
reaction. Coupling product was precipitated by acetone followed by centrifuged 10 min,
4000 rpm. The pellet was washed by acetone for three times and then was lyophilized.
The lyophilized coupling product was dissolved in DMSO. 10 fold excess of EHA

16

compared to ELP-Initiator was added to solution. The polymerization process was carried
out on 70 ℃ for two hours under atmosphere (N
2
or Ar
2
). The Final product was
collected in DI water by dialyzing out DMSO solvent and dicyclohexylurea (DCU)
formed as a by-product. Also, 2-Chloropropionic Acid, 2-Chloropropionyl Chloride and
Acryloyl Chloride are used as initiators. Conjugation and polymerization reaction were
confirmed using FTIR-ATR technique, Multi-Angle Light Scattering (MALS) and
Matrix-Assisted Laser Desorption/Ionization (MALDI).

17

CHAPTER 3
RESULTS
3.1 Confirmation of purity and correct molecular weight of selected ELPs by
SDS-PAGE

Fig.5: SDS-PAGE of selected ELP.
Fig.5: Absence of any other band confirms the purity of these ELPs. Lane 1: I96; Lane 2:
I48; Lane 3: I24; Lane 4: V96; Lane 5: V48; Lane 6: A192
As shown in Fig.5, there is only one band in all the lanes which means the purity of all the
samples is acceptable. The molecular weight of I96 and V96 are around 41kD while the
molecular weights of I48 and V48 are around 20kD, the one of I24 is around 10kD, and the
one of A192 is around 80kD. The results are consistent with expected values.

18

3.2 Determination of Concentration and Tt of ELPs by UV-Spectrophotometer
The concentrations and Tt of these selected ELPs were measured by
UV-Spectrophotometer. To measure the concentration of ELP, take V96 as an example,
the purified protein solution were diluted 10, 20, 50, 100, 200 folds separately and then
measured the absorbance at 280nm (Fig.6). The concentrations could be calculated by the
equation:
Conc (M) = (A280-A350)/1280

Fig.6: The wavelength scans of V96.
Then the same samples were run a temperature ramp to achieve their transition
temperatures as shown in Fig.7.

19

Fig.7: The Tm analysis of V96.
ELPs V48, V96 and I96 were characterized for their transition temperatures (Tt) in PBS
by UV-Vis Spectrophotometer (Beckman Coulter). The concentrations of V48 are
1.68mM, 1.12mM, 1.12mM, 0.85mM, 0.73mM, 0.37mM, 0.19mM, 0.08mM. The
concentrations of V96 are 0.87mM, 0.47mM, 0.20mM, 0.09mM, 0.05mM. The
concentrations of I96 are 0.72mM, 0.41mM, 0.20mM, 0.08mM, 0.05mM. Fig.8 displays
phase transition profile of ELPs V48, V96 and I96 with respect to their concentration.
When the concentrations of ELPs increase, the Tt of ELPs would decrease accordingly.
From Fig.8, the Tt of V48 is higher than that of V96 as they have the same guest residue
of valine. With the same guest residue, the more repeats of pentamers in the ELP, the
lower Tt the ELP has. Also, the Tt of I96 is lower than that of V96. With the same length
of ELPs, the more hydrophobic the guest residue is, the lower Tt the ELP has. For the

20

adhesive project, the proper Tt of the ideal ELP is around 34℃ as it’s the temperature of
body surface. The V96 is chosen as the material for the adhesive project because it could
get a Tt at a lower concentration and molecular weight of V96 is around 40kD which is
proper for the particle formation.

Fig.8: Compare the Tt difference between three different kinds of ELPs.
3.3 Thermal analysis of ELPs by Differential Scanning Calorimetry
All of the selected ELPs were also tested for their thermal transition by Differential
Scanning Calorimeter (DSC 8000 Perkin Elmer Inc.). 20ul of solutions with different
concentrations of ELPs were casted in aluminum pans (50ul) and then sealed by using
standard crimper. Different scans were run based on the type of ELPs. In general, the
temperature ramp was run from 4 ℃ to 80℃ with the rate of 1 ℃/min.

21

Fig.9 is a typical graph of the DSC result. The curve derived from the DSC result of V96
with a concentration of 1mM. The result demonstrates that the transion process is
endothermic. The onset temperature of the trantision is 26.5℃ while the peak temperature
of the transition is 27.4℃. Also, the ΔH of the transion process measured by the DSC
directly is 2.72J/g.

Fig.9: Transition phase of V96 measured by DSC.
In Fig.10, the transition temperatures measured by DSC were compared with those
measured by optical density. The differences of Tt between the results measured by those
two different ways were less than 1 ℃. So the results could be considered to be consistent.

22

(a)

(b)

23

(c)

(d)

24

(e)

(f)
Fig.10: Compare of the difference in transition temperatures measured by two different
ways.
Fig.11 is to compare the differences in ΔH (J/pentamer mol) of ELPs with Isoleucine as
guest residue but at different lengths. From Table.2, the ΔH (J/pentamer mol) of I48 has

25

no statistically significant difference from that of I24. However, there is a statistically
significant difference between ΔH (J/pentamer mol) of I24 and that of I96. It is the same
situation between I48 and I96.

Fig.11: Compare the different ΔH of ELP with Isoleucine at different lengths.

Tukey's Multiple
Comparison Test
Mean
Diff.
q Significant? P
< 0.05?
Summary
I48 vs I24 -150.9 0.5929 No ns
I48 vs I96 3159 9.98 Yes ***
I24 vs I96 3310 12.01 Yes ***

Table.2: Statistic analysis of the data presented in Fig.11

26

Fig.3-8 is to compare the differences in ΔH (J/pentamer mol) of ELPs with Valine as
guest residue but at different lengths. From Table.3, there is a statistically significant
difference between ΔH (J/pentamer mol) of V24 and that of V96.

Fig.12 : Compare the different ΔH of ELP with Valine at different lengths.
F test to compare variances
F,DFn, Dfd 33.58, 3, 4
P value 0.0054
P value summary **
Are variances significantly
different?
Yes
Table.3: Statistic analysis of the data presented in Fig.12
As expected, the ΔH (J/pentamer mol) should be consistent within the ELPs have same

27

guest residue. The experiment result didn’t consist with the hypothesis, however.
3.4 PSA-ELP conjugation confirmation by FTIR-ATR
PSA and ELP V96 chemical conjugation was carried out using several coupling agents in
separate ways. Conjugation was confirmed using FTIR-ATR technique using FTIR
Spectrometer (Bruker Vertex 80V).
As shown in Fig.13, the green line represents pure V96 while red line represents V96
conjugated with the initiator. The biggest difference between these two samples is that the
red line has two peak under 955cm
-1
and 1024cm
-1
, which indicates the exist of C-Cl
stretch while the green line has no peaks. This difference demonstrates that Cl has taken
place of the hydrogen atom at the N-terminus of V96, which means the coupling process
may work.

28

Fig.13: Compare the difference in absorbance peaks between pure V96 and V96 coupled
with initiator
As shown in Fig.14, the green line represents polymerized V96 while the red line
represents V96 conjugated with the initiator. The biggest difference between the two
samples is that the polymerized V96 lost the two peaks on 955cm
-1
and 1024cm
-1
, which
indicates the exist of C-Cl stretch in the conjugated V96. This difference demonstrates
that the EHA monomer replaces the Cl in the V96-initiator coupling product.

29

Fig.14: Compare the difference in absorbance peaks between V96-Initiator and
polymerized V96
3.5 Compare Tt of polymerized ELPs by UV-Spectrophotometer
Pure V96, V96-initiator and polymerized V96 were also run a temperature ramp with
UV-Spectrophotometer. As shown in Fig.15, the polymerized V96 has a much lower
transition temperature while V96-initiator conjugation has a slightly lower transition
temperature than that of the pure V96. The difference might be the result of the different
molecular weight. The initiator has tiny mass compared to pure V96 so the difference in
transition temperature is also slightly. However, the polymerized V96 might be linked
with huge amount of EHA monomers which affect the transition temperature a lot.

30

Fig.15: Tm analysis of V96, V96-Initiator and polymerized V96
3.6 Determination of accurate molecular weight of ELPs by MALDI
To measure the accurately molecular weight of the conjugated and polymerized products,
Matrix Assisted Laser Desorption/Ionization (MALDI) was applied to those samples.
From Fig.16, there are no big difference between (a) and (b). It’s hard to tell whether the
coupling success or not by this data. Take the FTIR-ATR result into consideration;
however, the coupling process should work as the C-Cl stretch appears. But it could be
concluded from Fig.16 (c) that the polymerization process failed as there’s no increase in
molecular weight. It is even lower than that of the V96-initiator conjugation. Combined
with the FTIR-ATR result, the initiator might fell off V96 instead being replaced by the
EHA monomer. This explains why the C-Cl stretch peak disappears in the polymerized
V96.

31

(a) (b) (c)
Fig.16: MALDI measurement of ELPs.
Fig.16: (a) Pure V96; (b) V96-initiator conjugation; (c) polymerized V96 with EHA

19768.2
39571.7
0
100
200
300

20000 25000 30000 35000 40000
m/z
39683.6
19817.0
0
100
200
300
400

20000 25000 30000 35000 40000
m/z
39574.1
19774.0
0
100
200
300
400
500

20000 25000 30000 35000 40000
m/z

32

CHAPTER 4
DISCUSSION
Protein-based polymers are comprised of repeating peptide sequences. In this study,
Elastic-Like Polypeptides consisted of pentamers (VPGXG)nY where the repeating unit
could be as few as two or as many as hundreds while the guest residue could be any of
the amino acids. The diversity and control of structure makes a library of different
polymers sharing certain same property: ELPs hydrophobically fold and assemble when
the temperature is rising, followed by the result of a clean phase separation(Urry, 1997).
As shown in Fig.6, the transition temperature (Tt) could be determined by
UV-spectrophotometer. The transition process representing a rearrangement of the
polymer structure could also be observed by differential scanning calorimetry (DSC).
The concentration and transition temperature of all the samples could be determined by
UV-Spectrophotometer as is shown in Fig.6 and Fig.7. From comparison of the transition
temperatures between different ELPs (Fig.8), V96 was chosen for further conjugation and
polymerization. As shown in Fig.10, the transition temperature measured by both
UV-Spectrophotometer and DSC are consistent. As stated in related research papers, the
change in entropy per pentamer during the transition process should be the same for all
ELPs with the same guest residue. However, the results from Fig.11 and Fig.3.8 were not
in accordance with this hypothesis. For isoleucine series shown in Fig.11, the ΔHs of

33

I48 and I24 had no statistically significant difference. ΔH of I96 was much lower than
those of I48 and I24. It is the same that ΔH of V96 is lower than that of V48. One
possible explanation is the length of the polymer chain might affect its thermal behavior.
It might be easier to induce the transition process when the chain is long.
In addition to the “soluble to insoluble” phase transition, ELPs also show ability to form a
crystalline phase-the glass transition process at higher temperatures, which might be an
exothermic system according to our DSC results.
In the history of the development on reversible bioadhesives, pressure sensitive adhesive
(PSA) polymers played an important role. In this project, primary aim was to develop a
novel type of reversible bioadhesive bandage material, the adhesive ability of which
would be dependent on the environment temperature. Its adhesiveness would reduce
when the temperature is lower than human skin temperature (34℃), which allows the
bandage to be removed with ease and lesser pain. To achieve this objective, effort were
made to grow an acrylate pressure sensitive adhesive polymer on the N-terminus of ELPs
by using the emulsion polymerization mechanism. V96 is obtained and purified from E.
coli bacterial culture, then used for the conjugation study in this project. The general idea
was to achieve ELP-PSA polymerization via coupling of the ELP with an initiator which
acts as propagating radical for further polymerization that is first followed by the addition
of the acrylate-based PSA monomer. In light of this, two different synthetic e routes were
chosen.

34

Fig.13 shows the comparison of pure ELP and ELP-initiator 2-Chloropropionic acid. We
found that conjugated V96 has two more peaks than pure ELP in the FTIR-ATR spectra,
which indicate the existence of C-Cl stretch. From Fig.14, the disappearance of those two
peaks in the polymerized V96 also indicates the absence of C-Cl stretch. Combined with
the MALDI result in Fig.16, there’s no big difference in molecular weight among the
three samples, which means the polymerization has failed. The coupling process might be
successful as the initiator is found in small amount as compared to V96 and therefore,
shows C-Cl peaks in FTIR-ATR. However, there’s the possibility that the C-Cl peaks
resulted from the residual in the solution instead of V96 chain. On the other side, the
polymerization process failed as there was expected to be multiple peaks with different
peaks, which should be normal distributed. The disappearance of C-Cl stretch in
polymerized ELP might result from falling off of the initiator from V96 during the
polymerization process. For Fig.15, one possible explanation for the differences in
transition temperature of these three samples is that the remained chemical agents might
affect polymers’ thermal behavior.

35

CHAPTER 5
FUTURE DIRECTIONS
In the study of the properties of Elastin-Like Polypeptides, a better knowledge of the
physical chemistry of the transition process is quite beneficial to understanding the
thermal behavior of ELPs. In present study, I24, I48, I96 and V48, V96 were chose for
such measurements. As the results conflict with existing opinion, further experiments are
needed to arrive to a better conclusion. We also feel that the alanine series of ELPs should
also be included in the experiments. On one hand, it provides another group to compare,
which could increase reliability of the results. On the other hand, V96, I96 and A96,
which are of the same length, could be compared to find out the effect of different guest
residue on the ELPs’ physiochemical properties.
In present study, emulsion polymerization method is being utilized for elongation ELP
with EHA monomers. Several initiators and agents have been tried for the polymerization,
including 2-Chloropropionic Acid, 2-Chloropropionyl Chloride and Acryloyl Chloride as
well as RAFT agents. To date, these samples have not been characterized by MALDI
mass spectrometry. Also, Multi Angel Light Scattering (MALS) should also be taken into
consideration as a mean of molecular weight measurement. So far, the difference between
the pure ELP and ELP-initiator conjugate could not be observed by MALS. However, its
amount could be small and below the detection limits of the currently used method.

36

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Gholap, V. (2012). Elastin Like Polypeptides as Thermalresponsive Modulators in
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Creator Wang, Jiawei (author)

Core Title Physical chemistry and polymeric modification of elastin-like polypeptides

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 04/30/2014

Defense Date 03/20/2014

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