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Genetic engineering of thermally sensitive elastin-like polypeptide and its expression in HEK 293 cells

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Genetic engineering of thermally sensitive elastin-like polypeptide and its expression in HEK 293 cells

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GENETIC ENGINEERING OF THERMALLY SENSITIVE ELASTIN-LIKE
POLYPEPTIDE AND ITS EXPRESSION IN HEK 293 CELLS

by

Sejal Parakh

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)

August 2010

Copyright 2010 Sejal Parakh
ii

ACKNOWLEDGEMENTS
I would like to thank my guide Dr. Andrew MacKay for his guidance, insight and
encouragement throughout the project. I would also like to express my gratitude towards
my thesis committee members Dr. Sarah Hamm-Alvarez and Dr. Curtis T. Okamoto for
their guidance, advice and time. I greatly appreciate all the support and guidance from my
colleagues in the MacKay Laboratory especially Siti Mohd Janib, Vinod Valluripalli,
Martha Pastuszka, Wan Wang, Aarti Jashnani and Suhaas Aluri. I would also like to
thank my family and friends for their encouragement and support throughout my master’s
program. Finally, I would like to show my gratitude and appreciation to the USC School
of Pharmacy for supporting this project.

iii

TABLE OF CONTENTS
ACKNOWLEDGEMENTS ii
LIST OF TABLES v
LIST OF FIGURES vi
ABBREVIATIONS viii
ABSTRACT x
CHAPTER 1: INTRODUCTION 1
CHAPTER 2: BACKGROUND 3
2.1 Elastin-like polypeptides 3
2.2 Enhanced Permeability and Retention Effect 6
2.3 Endoplasmic Reticulum Signal 7
2.4 N-glycosylation Process 10
2.5 Aim of the project 12

CHAPTER 3: METHODS 16
3.1 Miniprep/Maxiprep/Gigaprep 16
3.2 Recursive Directional Ligation 17
3.3 Agarose Gel Electrophoresis 18
3.4 Gel Extraction 19
3.5 Ligation 20
3.6 Transformation 20
3.7 Inverse Transition Cycling for protein purification 21
3.8 SDS PAGE 22
3.9 Cell Culture 22
3.10 Transfection 23
iv

CHAPTER 4: MATERIALS AND PROCEDURES 24
4.1 Design of various constructs using Lasergene 24
4.2 General procedure to insert desired gene sequence into a vector 24
4.3 Construction of ELP-Alanine Library in pET25b+ vector 25
4.4 Recombinant Synthesis of SP1 into pcDNA3.1+ vector 26
4.5 Recombinant Synthesis of A
96
and A
192
into pcDNA3.1+ vector 28
4.6 Recombinant synthesis of SP3-A
192
into pcDNA3.1+ vector 29
4.7 Protein Purification by ITC 29
4.8 Measurement of Tt using Beckman Coulter DU 800
UV-vis spectrophotometer 31
4.9 SDS PAGE 31
4.10 Cell Culture and transfection method 31
4.10.1 Starting HEK 293 Cell Cultures from Frozen Stocks 32
4.10.2 Passaging HEK 293 cells 33
4.10.3 Transfection of different plasmids into HEK 293 cells 33
CHAPTER 5: RESULTS 36
5.1 ELP VPGAG Library constructs 36
5.2 Transition Temperature for ELP A
192
at different concentrations 38
5.3 Determination of protein expression of A
96
and SP1- A
192
39
5.4 Transfection of HEK 293 cells using different pcDNA3.1+ constructs 40
5.4.1 Liposome as transfection agent 40
5.4.2 L-PEI as transfection agent 42

CHAPTER 6: CONCLUSION 48
CHAPTER 7: FUTURE DIRECTIONS 50
REFERENCES 51
v

LIST OF TABLES

Table 1: Typical Signal Sequences showing the importance of
positively charged (red), negatively charged (green), hydrophobic
(yellow) and hydroxylated amino acids (blue) for entry or exit
into a specific cell organelle 8

Table 2: Amino Acid Sequences of ER Signal Peptides in
Three Eukaryotic Proteins 15

Table 3: Expected band size for respective length of ELP 37
Table 4: Samples introduced in each lane for SDS PAGEshown in Figure 14 40
vi

LIST OF FIGURES
Figure 1: Phase transition of ELPs 6

Figure 2: Types of ER SS 8

Figure 3: The Signal Recognition Particle 9

Figure 4: Role of ER SS and SRP to transfer ribosomes to the ER membrane 10

Figure 5: Transfer of precursor oligosaccharide from dolichol to amino
terminus of Asn 11

Figure 6: N-linked glycosylation in the ER and the Golgi apparatus 12

Figure7: Formation of N-glycosylated ELP nanoparticles with
encapsulated drug above Tt from its monomers (below Tt ) 13

Figure 8: Schematic presentation of the N-glycosylation+ELP construct 14

Figure 9: Recursive Directional Ligation Technique 18

Figure 10: ITC method for protein purification 21

Figure 11: ELP VPGAG library 36

Figure 12: SP1 sequence consisting of ER SS and 8 NGS glycosylation 38
units is shown in the block.

Figure 13: Tt of A192 at different concentrations 39

Figure 14: Determination of expression of glycosylated ELP or
only ELP in HEK 293 cells using SDS PAGE 39

Figure 15: FITC and DIC images of GFP and EGFP transfection into
HEK 293 cells using Liposome as transfection agent (4.10.3.A) 41

Figure 16: FITC and DIC images of GFP and EGFP transfection into
HEK 293 cells using L-PEI as transfection agent (4.10.3.B) 43

vii

Figure 17: FITC and DIC images of GFP and EGFP transfection into
HEK 293 cells using L-PEI as transfection agent (4.10.3.C) 45

Figure 18: FITC and DIC images of GFP and EGFP transfection into
HEK 293 cells using L-PEI as transfection agent (4.10.3.E) 47

viii

ABBREVIATIONS
A
6
(VPGAG)
6

A
12
(VPGAG)
12

A
24
(VPGAG)
24
A
48
(VPGAG)
48
A
96
(VPGAG)
96

A
192
(VPGAG)
192
Asn/N Aspargine
CIP Alkaline phosphatase, Calf intestine
DIC Differential Image Contrast
DMSO Dimethyl sulfoxide (DMSO)
DNA Deoxyribonucleic acid
E. coli Escherichia coli
EGFP Enhanced green fluorescent protein
ELP Elastin-like polypeptides
ER Endoplasmic Reticulum
FBS Fetal Bovine Serum
FITC Fluorescein Isothiocyanate
GFP Green fluorescent protein
ITC Inverse transition cycling
LB Luria-Bertani
ix

L-PEI Linear Poly(ethylenimine)
M.W. Molecular weight
NEB New England Biolabs
PBS Phosphate buffer saline
RDL Recursive directional ligation
RE Restriction Enzyme
rRNA Ribosomal ribonucleic acid
RT Room Temperature
SAM S-adenosyl Methionine
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Ser/S Serine
SP1 ER SS+8 NGS
SP1-A
192
ER SS+8 NGS+ A
192

SP2 A
n
(n = 6, 12, 24, 48, 96, 192)
SP3 ER SS+1 NGS
SS Secretion Signal
SRP Signal Recognition Particle
TB Terrific broth
Thr Threonine
T
t
Transition temperature
x

ABSTRACT
Substantial efforts have been to design drug carriers for targeted drug delivery to
tumor sites in order to spare the normal tissue from the hazardous effects of
chemotherapeutic agents and achieve better therapeutic efficacy [2, 4, 37, 39]. One
promising approach to reduce chemotoxicity would be to conjugate or encapsulate these
potent drugs into nanometer (10-400) sized particles and trigger their release at tumor
sites [12, 13, 20]. However, nanoparticulates frequently accumulate in the liver, which
leads to high local concentrations of these toxic drugs. This accumulation reduces
delivery of the appropriate dose of drugs to the site of action and can generate
hepatotoxicity.
The aim of this project is to design a nano drug carrier that extends plasma
circulation, permits greater tumor accumulation, and reduces hepatotoxicity, which is
based on the enhanced permeability and retention effect. To achieve this goal, a novel
approach to produce N-glycosylated peptide nanoparticles has been explored.
Elastin-like polypeptides are repeated pentameric peptides VPGXG exhibiting a
characteristic inverse phase transition temperature T
t
, above which they form aggregates
from a monodisperse solution. It is hypothesized that on triggering the phase transition of
an N-glycosylated-ELP-drug conjugate, it will form nanostructures with insoluble ELP-
xi

drug at the core shielded with soluble N-linked carbohydrates. For this purpose, a library
of ELP VPGAG consisting of 6, 12, 24, 48, 96, 192 pentamers was constructed. This was
followed by biosynthesis of several constructs comprising of ELP A
96
and A
192
with
Endoplasmic Reticulum Signal and N-glycosylation sequences using recombinant DNA
technology.
Expression of these constructs and GFP plasmid was optimized into mammalian
HEK 293 cells using L-PEI as transfection agent. Successful expression of GFP protein
alone and its co-transfection with N-glycosylation + ELP A
192
and A
96
sequences was
evaluated using fluorescence microscopy. However, N-glycosylated ELP or ELP (A
192

and A
96
) did not exhibit any obvious phase transition in the medium collected before and
after HEK293 cell lysis. Thus, expression of N-glycosylated ELP or ELP protein could
not be determined.
1

CHAPTER 1
INTRODUCTION
Cancer is a class of diseases characterized by an uncontrolled growth of abnormal
cells in the body. These rapidly growing malignant cells can invade adjacent tissues or
metastasize to other organs of the body through blood or lymph.
The change from a normal cell into a tumor is a multistage process as a
consequence of several factors such as [45]:
 Heredity: Individual’s genetic factors
 Mutation due to the exposure to carcinogens: Physical – UV rays, Chemical –
Tobacco
 Infections - Human Papilloma Virus / H.pylori
 Hormonal Imbalances [29]
 Lifestyle, diet and alcohol intake [7, 10]
 Ageing
According to the American Cancer Society, over 7.5 million deaths occurred
worldwide due to cancer. Cancer is responsible for one in eight deaths in the world. It
also accounts for 20,000 deaths on a daily basis. The main types of cancer leading to
overall cancer mortality each year are [45]:

2

 lung (1.3 million deaths/year)
 stomach (803 000 deaths)
 colorectal (639 000 deaths)
 liver (610 000 deaths)
 breast (519 000 deaths)
Although specific therapies have improved survival within some subtypes of
cancer over the last 30 years, the continued prevalence of cancer mortality and morbidity
demands constant innovation. Chemotherapy is one of the most common treatments used
to manage this disease. Substantial research has been made to overcome the toxicities
caused by the use of potent chemotherapeutics. One widely explored approach is the
development of drug carriers that improve the selectivity of drugs to tumors and reduce
exposure to sites of toxicity.
ELPs are smart biopolymers that phase separates out from aqueous solution above
their characteristic transition temperature (T
t
). We hypothesize that the attachment of
bulky sugar groups to this polypeptide can form micelles with the ELP and drug at the
core coated by N-glycosylated sugars, which can be induced to assemble simply by
raising the temperature. Protected by humanized sugars, these glycosylated nanoparticles
might circulate for extended periods in the body, be degraded upon cellular uptake, be a
platform for triggered drug release, reduce drug exposure to normal tissues, and achieve
better therapeutic activity [5].
3

CHAPTER 2
BACKGROUND
2.1 Elastin-like Polypeptides
Elastin-like Polypeptides (ELPs) belong to the class of smart polymers that is
responsive to temperature stimuli at a characteristic transition temperature T
t
. This
property of the ELPs is a manifestation of the unique repeated amino acid pentameric
units of (VPGXG)n, where X represents any amino acid other than Proline and n is the
length of the pentameric repeats [9]. The polymeric ELP exists as a helical structure of β-
turns called a β-spiral. Recurrent β-turns due to the presence of PG dipeptide acts as a
spacer for hydrophobic interactions between the helical turns. Insertion of Proline as a
guest amino acid residue X would interrupt with the formation of helical structure of ELP
VPGXG. This, in turn, would alter the hydrophobic connections required for formation of
ELP aggregates on raising the temperature [25, 42, 43].
The repetitive ELP phenomenon was inspired from W4 domain of human
tropoelastin that consisted of repetitive VPGG, VPGVG, and APGVGV peptides [18, 38,
40]. These hydrophobic repeats exhibited reversible coacervation at 150 mm NaCl, 37°C,
and pH 7–8 [18, 44]. It was found that formation of β-turns due to the presence of PG
dipeptide helped in aggregation of these polymers [18, 38, 40, 44].
4

Below their T
t
, ELPs are soluble in aqueous solutions; however, after crossing the
characteristic T
t
they collapse to form hydrophobic aggregates. T
t
can be modulated by
several factors, including the guest amino acid residue (X), concentration, ionic strength,
chain length (n), and pH. For instance, T
t
increases with an increase in the hydrophobicity
of X but is inversely proportional to the chain length and concentration of ELP [9, 31].
Being a biopolymer, ELPs are biocompatible and biodegradable [9, 12].
ELPs can be genetically engineered and expressed in mammalian and bacterial
cells. Genetic engineering allows regulation of ELP T
t
through modification of several
characteristics of ELPs like its length or guest residue [9, 31, 42]. ELP gene can be easily
fused with other genes of interest that may help in expression of any desired protein
through recombinant DNA technology. This opens numerous opportunities such as
detection of protein expression in the body by HexaHis tagging of ELP [22, 36] or
formation of fusion protein like ELP and human IL-1 receptor antagonist IL-1ra-ELP
[19] or understands the function of the protein.
T
t
allows an easy, rapid and economical method for protein purification called
Inverse Transition Cycling technique. On triggering phase transition, ELP protein
aggregates which is filtered or centrifuged. Due to the reversible phase transition property
of ELPs, the pelleted ELP protein is re-solubilized after removal of debris in supernatant
by dissolution in an aqueous solution below its T
t
. This ITC process enables protein
purification of ELPs alone or ELP-fusion proteins at large and small purification scales
without denaturation of fused protein. It can be used to determine unknown binding
5

partners to the protein of interest (antibody, receptor, etc) fused with ELP followed by the
characterization of its binding and rate constants using spectroscopic techniques or
competitive immunoassays [15, 32, 34, 41].
T
t
allows self-assembly of ELP diblock copolymers into nanostructures [10, 46].
AB diblock copolymer of ELPs can be biosynthesized wherein A and B pentameric
blocks consist of different guest residue X. Formation of self-assembled nanostructures
like micelles of such ELP diblock peptides is governed by the guest residue, length,
molecular weight, ELP concentration and the amphiphilic nature of the diblock polymer.
These factors tune the overall T
t
of the block copolymer such that a monodisperse
solution exists below the T
t
of both ELP blocks. As the temperature rises, self-assembly
into spherical micelles results when the low T
t
block is surpassed. At this stage, the
micelle would consist of an insoluble hydrophobic ELP block (low T
t
) at the core
surrounded by a more soluble hydrophilic ELP block (high T
t
). The hydrophilic-
hydrophobic ratio and length of ELP block copolymer controls the size of the micelle [8,
10, 46]. Similar incorporation of drugs or radiolabeling agents achieved through
chelation, conjugation or encapsulation to ELP moiety can form micelles or fusion motif
structures on manipulation of the T
t
[9, 13].
6

Fig.1 Phase transition of ELPs: Formation of ELP aggregates (right) on heating above
its Tt / salt addition and reversible conversion into soluble phase (left) on cooling.

2.2 Enhanced Permeability and Retention Effect
Enhanced accumulation of lipid- or polymer- conjugated drug is reported over
free drug in tumor tissue than in normal tissue due to the enhanced permeability and
retention (EPR) effect. This phenomenon is attributed to the abnormal vascular
architecture and decreased lymphatic drainage in solid tumors. This concept of EPR is
becoming a gold standard in anti-tumor drug delivery [16, 26, 28].
To meet the growing and high demand of nutrient and blood supply of the rapidly
dividing tumor cells, blood vessel growth (angiogenesis) is induced by secretion of
several growth factors like VEGF. Hypervasculature, increased angiogenesis and
haphazard blood flow due to impaired functional receptors contribute in transition of
harmless cells into a large tumor and metastasis [14, 26, 27].
Increased extravasation of macromolecules from tumor blood vessels to tumor
tissues was contributed by large intercellular openings in the vessels (approximately
4.7μm), lack of smooth-muscle layer and elevated levels of vascular permeability factors
such as bradykinin, nitric oxide, peroxynitrite, metalloproteinases, VEGF, prostaglandins
7

and cytokines. Retention of macromolecules in tumor tissues is observed due to slow
venous return and higher molecular weight than that required for renal clearance of these
bulky molecules (approximately > 40 kDa) [14, 26, 27].
EPR effect has facilitated targeted delivery of lipid-and polymer- conjugated
drugs such as SMANCS [polystyrene-co-maleic acid conjugated neocarzinostatin] and
PK-1 [synthetic N-(2-hydroxypropyl) methacrylamide copolymer-doxorubicin conjugate]
to tumor sites [23, 28]. The drug carrier chosen should not be immunogenic or antigenic
and should fall in the molecular range of 15,000-70,000 to deliver anti-cancer
therapeutics utilizing EPR phenomenon [30].

2.3 Endoplasmic Reticulum Signal Sequence (ER SS):
The function of the signal sequence is to transport proteins from the nucleus to the
cytosol, Golgi apparatus to the ER or cytosol to the ER, chloroplasts, mitochondria and
peroxisomes. There are two types of signal sequences observed in proteins as shown in
the Fig.2. The first type consists of typically 15-60 continuous stretch of amino acids. It
can be located either at the end or at the interior of the polypeptide chain. The other type
of the signal sequence involves the formation of a signal patch due to the assembly of the
intermittently positioned specific amino acids [1].
8

Fig.2 Types of ER SS. (A) Continuous stretch of Amino acid SS located at the end of the peptide.
(B) Signal Patch formed due to the adjacent placement of the appropriate amino acid sequence
[1].
Signal sequence destined to a specific organelle possesses few characteristic
features. Focusing on the ER SS, it is usually present at the N-terminus of the protein
composed of about 6-12 hydrophobic amino acid residues often preceded by one or more
basic amino acids and it is cleaved by the signal peptidase at the rough ER surface [1,
24].
Table 1: Typical Signal Sequences showing the importance of positively charged (red),
negatively charged (green), hydrophobic (yellow) and hydroxylated amino acids (blue)
for entry or exit into a specific cell organelle [1].

9

The hydrophobic core of the ER SS is crucial for its binding with the signal
receptor particle (SRP) which allows recognition of different ER SS by the sorting
receptor irrespective of composition or length of the ER SS.
The ER SS binding site on the SRP has a hydrophobic pocket made of
methionines whose unbranched, flexible side chains readily holds SS of varying
sequences and shapes.

Fig.3: The Signal Recognition Particle (A) A mammalian SRP made of 6 protein
subunits and 1SRP RNA unit that binds to rRNA.(B) A crystal structure of SS binding
domain of a bacterial SRP subunit. The hydrophobic methionine domain (gray) is crucial
for interaction with hydrophobic parts of different SS [1].

Translation process of the protein is paused after the SRP binds to the ER SS and
the ribosome. The SRP receptor complexes with ribosome bound SRP and directs it to
the translocator. Transfer of the polypeptide across the lipid bilayer is facilitated through
this translocator moiety. Hydrolysis of GTP by P54 subunit of SRP and α subunit of the
SRP receptor is essential for the attachment of the ribosome to the ER and forwarding the
polypeptide into the ER. It is reported that secretory proteins composed of more than 100
amino acids undergo a cotranslational process. It means that such proteins are transported
into the ER lumen while they are being translated.
10

Fig.4: Role of ER SS and SRP to transfer ribosomes to the ER membrane [1]
After successful binding of the ER SS, the polypeptide is transferred through a
pore opened by the translocator. The SS is cleaved off by the signal peptidase followed
by its release from the pore and finally, degradation by the ER proteases. The
translocated protein is released into the ER lumen only after the passage of its C-terminus
through the membrane [1, 24].

2.4 N-glycosylation process
Protein glycosylation is one of the major functions of the ER. Proteins with an
attachment of an oligosaccharide to the side-chain NH
2
group of an aspargine/N present
in the consensus sequence Asn-X-Ser/Thr (X-any amino acid other than Proline) are
called as N-linked sugars or N-glycosylated protein or N-glycoproteins.
As soon as the polypeptide enters the ER lumen, a precursor oligosaccharide (14
sugars) is transferred from dolichol to the amino terminus of Asn in Asn-X-Ser/Thr
catalyzed by oligosaccharyl transferase (Fig.5). The energy is derived from the
pyrophosphate bonds present between the dolichol and 14 sugar moiety [1, 24].
11

Fig.5: Transfer of precursor oligosaccharide from dolichol to amino terminus of Asn
[1]
N-linked oligosaccharides can form either high-mannose oligosaccharides or
complex oligosaccharides. The former contains only two N-acetylglucosamines and
several mannose residues. Complex oligosaccharides are formed as a result of trimming
certain glucose and mannose residues from the original N-linked oligosaccharide added
in the ER and then further addition of sugars in the Golgi apparatus. Stepwise removal of
3 glucose and 1 mannose is catalyzed by glucosidase I, II and mannosidase in the ER
respectively. This moiety is then subjected to Golgi mannosidase that eliminates
additional 3 mannoses and N-acetylglucosamine transferase I adds an N-
acetylglucosamine. After further removal of 2 mannoses by Golgi mannosidase II, the
bond between the two N-acetylglucosamines in the core becomes resistant to attack by
endoglycosidase (Endo H). Thus, treatment with Endo H enzyme helps to distinguish
high-mannose oligosaccharides from complex ones. The final step in the formation of
12

complex N-linked oligosaccharides occurs in the cisternal compartments of the Golgi
apparatus. The ultimate product has additional N- acetylglucosamines, galactoses and
negatively charged sialic acids (Fig.6).

Fig.6: N-linked glycosylation in the ER and the Golgi apparatus [1].

2.5 Aim of the Project
The focus of this project is to design N-glycosylated elastin-like polypeptide
using recombinant techniques. Along with the making of appropriate plasmid constructs,
expression of the glycoprotein in mammalian HEK 293 cells is also an objective.
13

Fig.7: Formation of N-glycosylated ELP nanoparticles with encapsulated drug above
T
t
from its monomers (below T
t
)

Rationale of the ER SS + NGS + ELP construct:
One of the main purposes is to achieve N-glycosylation of protein with
humanized glycoforms to avoid incompatibilities with human use of this drug carrier. It
has been reported that N-linked oligosaccharides are formed on the NH
2
terminus of the
aspargine amino acid of Asn-X-Ser/Thr consensus sequence [1, 3, 24]. Thus, for this
project 8 Asn-Gly-Ser units, each separated by a glycine amino acid was chosen. Glycine
was selected as it is the smallest of all the amino acids (minimum stearic hindrance to the
bulky sugar groups), non-chiral, neutral and fits well into hydrophobic as well as
hydrophilic environment.
14

Fig.8: Schematic presentation of the N-glycosylation+ELP construct
Since sugar addition to the proteins begins in rough endoplasmic reticulum (ER),
there was a need to direct the desired peptide sequence into the ER. This function is
carried out by ER signal sequence that directs any attached amino acid sequence to the
ER lumen for various modifications including post-translational modifications. To satisfy
this need, an ER SS was obtained from Pre-IgG light chain which had hydrophobic
amino acid residues and a cleavage site between cysteine and aspartic acid as shown in
Table 2 [1, 24].
ELP VPGAG was selected with alanine as the guest residue as its appropriate
length could exhibit phase transition near the body temperature.
15

Table 2: Amino Acid Sequences of ER Signal Peptides in Three Eukaryotic Proteins
[24] Blue: hydrophobic residues, Red: Cleavage by signal peptidase
Protein ER SS Peptide
Pre-IgG light
chain
Met-Asp-Met-Arg-Ala-Pro-Ala-Gln-Ile-Phe-Gly-Phe-Leu-Leu-Leu-
Leu-Phe-Pro-Gly- Thr-Arg-Cys ↓ Asp . . .
Preproalbumin Met-Lys-Trp-Val-Thr-Phe-Leu-Leu-Leu-Leu-Phe-Ile-Ser-Gly-Ser-
Ala-Phe-Ser ↓ Arg . . .
Prelysozyme Met-Arg-Ser-Leu-Leu-Ile-Leu-Val-Leu-Cys-Phe-Leu-Pro-Leu-Ala-
Ala-Leu-Gly ↓ Lys . . .

16

CHAPTER 3
METHODS
This section briefly describes the principles behind the various experiments performed.
3.1 Miniprep/Maxiprep/Gigaprep
This experiment is used to isolate circular plasmid DNA from bacterial cells. It is
based on alkaline lysis method developed by Birnboim and Doly [6].
Cell membrane is destabilized due to chelation of divalent cations like calcium
and magnesium by EDTA on resuspension of the pelleted cells after an overnight
incubation in the selected medium. Further loosening of the cell wall occurs due to the
presence of highly alkaline conditions contributed by NaOH and SDS. NaOH linearizes
and separates the chromosomal DNA into single strands but denatures plasmid DNA to a
certain extent only. Renaturation of circular DNA, precipitation of ssDNA and removal
of SDS (in the form of potassium dodecyl sulphate) is effected by the addition of
potassium acetate. Plasmid DNA is relatively small and supercoiled as opposed to larger
and less supercoiled chromosomal DNA of bacteria. Due to this, the latter is selectively
precipitated along with other cellular proteins leaving the plasmid DNA in the solution on
addition of potassium acetate. DNA is precipitated from the solution using isopropanol or
ethanol and resuspended in TE buffer or water.
17

3.2 Recursive Directional Ligation
Recursive Directional Ligation is a recombinant technique to synthesize
biopolymers. This technique involves joining of DNA segments sequentially such that the
length of the ligated segments grows geometrically in each step [9]. It is suitable for the
construction of long repetitive polypeptides like ELPs. Hence, effects of various
parameters like molecular weight, guest residue, type and length of polymer,
concentration on the phase transition temperature of the ELP can be studied easily.
RDL method consists of digestion of two aliquots of the vector with different set
of restriction enzymes (RE) such that only ELP and ELP+vector sequences are obtained
which can then be ligated to obtain the desired ELP length [33]. Depending on the
desired size of ELP, the vector may contain different ELP lengths. For example, if 24
ELP pentamer is to be biosynthesized, then only one vector with 12 pentamers of ELP is
subjected to one round of RDL digestion. This oligomerization process helps to maintain
the head-to-tail orientation of the insert, restriction sites of the REs used on the product
vector and selectively ligate only the ELP oligomer.
Specific features of the chosen recognition sites make RDL an effective technique
for building ELP library. The REs should not produce cohesive overhangs on the same
vector but are compatible with those on the cloning vector. It is important for the REs on
the cloning vector to be unique. After the ligation of the polypeptide sequence, there
should be no internal disruption due to either of the REs on the final pentameric repeats
[9].
18

Fig.9: Recursive Directional Ligation Technique
3.3 Agarose Gel Electrophoresis
This is one of the easiest ways to separate and analyze the DNA or RNA
fragments based on their sizes. It is useful for diagnostic procedure after DNA digestion
with enzymes, to quantify, isolate or visualize a specific DNA band, analysis of PCR
products and DNA fingerprinting.
The phosphate backbone of the DNA imparts it a negative charge at a neutral pH
which allows its migration from the negative to the positive pole on agarose matrix under
the influence of the electric field. Agarose is made of long unbranched, uncharged
carbohydrate chains resulting in a porous lattice, through the holes of which the DNA
makes its way. Smaller molecules can maneuver easily through the pores than the larger
ones, thus running faster and farther on the gel. Low percent gels show good resolution
for larger DNA fragments but are fragile. Small fragments are better separated using high
percent gels. This is the basis of DNA separation according to its size. Agarose gel is
19

immersed in Tris-acetate EDTA buffer which is suitable for larger DNA resolution.
However, it requires lower voltage and more time. The progress of the gel can be
monitored using loading buffer in the sample. The buffer gives color and density to the
loaded sample and moves towards the positive pole as it is negatively charged. Different
DNA bands are visualized on a U.V. trans-illuminator after staining with SYBR Safe dye.
DNA size markers help to identify the band size of the DNA under analysis.

3.4 Gel Extraction
This technique is used to isolate DNA fragment from an agarose gel following gel
electrophoresis. Three basic steps are involved in this extraction process : washing,
binding and elution of DNA from the excised gel portion.
It is based on selective adsorption of DNA owing to the presence of chaotropic
salts followed by its elution in low ionic strength buffer or deionized water. These salts
destroy the relatively ordered structure of water molecules covering the DNA rendering it
water-insoluble. This increases its affinity to bind to the silica membranes of the spin
column. Proteins, metabolites and other impurities are washed off using buffers with a
high concentration of chaotropic salts like ethanolic buffers. Additionally, chaotropic
salts are removed from the silica membrane without affecting the DNA-silica bonds.
Rehydration with an aqueous buffer or deionized water breaks DNA-silica bonds and
elutes pure DNA.

20

3.5 Ligation
This is the final step in construction of a recombinant DNA molecule wherein the
DNA to be cloned is joined to the vector in presence of DNA ligase. Ligase catalyzes the
formation of two covalent phosphodiester bonds between 3’ hydroxyl ends of one
nucleotide with the 5’ phosphate end of another. It is an energy driven process requiring
ATP. Thus, blunt or sticky ends of the dsDNA strands can be ligated to achieve
successful insertion of a gene, DNA repair or replication. Usually, for successful
ligations, the optimal enzyme temperature used is 25°C for T4 DNA Ligase.

3.6 Transformation
Introduction of a foreign DNA such as plasmid into the bacterial cells is known as
transformation. This helps to amplify the desired genetic material in order to get large
amounts of it. Bacterial cells that are capable of undergoing transformation are termed as
competent cells. In order that only the cells containing plasmid with the gene of interest
are transformed, plasmids contain an antibiotic resistant gene. Thus, cells lacking the
plasmid will not grow. Heat shock is one of the methods to transfer the plasmids into the
bacterial cells. The exact mechanism is not fully understood yet. Initially, the cells are
made leaky with the help of calcium chloride so that the DNA enters the bacteria. Heat
shocking at 42
o
C seals the holes formed in the cell wall and membrane. The cells are then
plated on selective medium agar plates.
21

3.7 Inverse Transition Cycling for protein purification [34]:
This is a protein purification method for ELPs taking advantage of their specific
inverse phase transition temperature. Firstly, the protein is expressed in bacterial cells
which are lysed in order to release ELPs. To ease the purification process and reduce the
T
t
, the solution is mixed with salts such as sodium chloride or ammonium sulphate at
37
o
C. As a result of this, ELPs form aggregates indicated by turbidity of the solution.
The ELP aggregates are pelleted out and resolubilized in PBS below its T
t
so that it
remains in the solution. This process is repeated atleast twice to obtain pure ELP. The T
t
of the protein

is determined using DU800 UV-vis spectrophotometer. SDS-PAGE
confirms the purity of the protein.

Fig.10: ITC method for protein purification
22

3.8 SDS PAGE
Sodium dodecyl sulphate polyacrylamide gel electrophoresis is a common method
used to resolve protein mixtures according to their sizes or molecular weight. It helps to
identify proteins or disulfide bonds, determine the protein size and purity.
SDS being an anionic detergent denatures secondary and tertiary structures of
proteins acquiring primary conformation coated with negatively charged sulphate groups.
This ensures that the protein separation occurs solely on the basis of the molecular weight
and not the order of protein folding. Approximately, 1.4g SDS binds to 1.0g protein
which can vary from 1.1-2.2 g SDS/g protein. Polyacrylamide gel offers resistance to the
movement of the negatively charged proteins due to its three-dimensional mesh-like
structure. Thus, like an agarose gel, smaller molecules make their way faster than the
larger one and run farther than the latter. This leads to separation of proteins only on their
size basis. Finally, the bands are visualized by staining methods like Copper Chloride or
Coomassie Blue.

3.9 Cell Culture
HEK cell line is derived from human embryonic kidney cells that expresses
transforming Adenovirus gene 5 from both the ends of viral genome. The various reasons
for which this cell line was chosen for the experiments are as follows:
 Quick and easy reproduction
 Stable and transient transfections possible
23

 Simple to handle and maintain
 High transfection efficiency and protein production
 Allows post-translational modifications with both mammalian and non-
mammalian nucleic acids

3.10 Transfection
Transfection means the introduction of foreign material like genetic material or
proteins into eukaryotic cells with the help of transfection agents, virus or other means.
Several methods are used to allow the uptake of the desired matter either through opening
of transient pores in or fusion with the cell membrane.

24

CHAPTER 4
MATERIALS AND PROCEDURES
4.1 Design of various constructs using Lasergene:
DNASTAR Lasergene was used to design the constructs containing ELP library
VPGAG, SP1, SP2 and SP3. SP1, SP2 and SP3 sequence was modified to enable the
insertion of ELP VPGAG of any length.

4.2 General procedure to insert desired gene sequence into a vector
All the enzymes and buffers used were obtained from New England Biolabs® Inc.
The basic steps followed to build the different constructs in any vector involved:
1. Digestion of the Vector A (containing the insert) as well as the vector B with
same unique restriction enzymes to obtain the desired insert and open up sticky
ends respectively for insertion.
2. Ligation of the excised oligonucleotide (from Vector A) into the Vector B.
3. Transformation of the ligation mixture into competent cells like TOP10
TM
cells on
TB-agar plates containing ampicillin or kanamycin (100mg/ml).
4. The colonies obtained are then subjected to a Diagnostic Digest with two unique
REs flanking the desired insert on Vector B.
5. On confirmation of the preferred band size from the Agarose gel electrophoresis,
the plasmid is sent for DNA sequencing.
25

4.3 Construction of ELP-Alanine Library in pET25b+ vector
Cassette Preparation: Forward and Reverse primers (100pmol/μl) encoding for ELP
VPGAG were annealed in the presence of ligase buffer. The reaction mixture was placed
in the heat block for 2 mins at 95°C and allowed to cool at room temperature for 3 hrs.
Vector preparation: About 1.5μg pET25b+ vector was digested with XbaI and BamHI at
37°C for 3hrs. CIP treated vector was then heat deactivated at 75°Cfor 10 mins. Spin
purification and elution was done in sterile water.
Ligation: Oligo:Vector of ~10-20:1 was ligated with the help of ligase buffer and T4
DNA ligase at RT for 2 hrs.
Transformation: The ligation mixture was then transformed into E. coli TOP10 cells and
plated onto ampicillin plates.
Diagnostic Digest: XbaI and BamHI in presence of NEB 3 were used to confirm the A
6
in the vector followed by gene sequencing.
The ELP-Alanine library was synthesized using RDL technique.
RDL A enzymes: AcuI and BssHII, NEB 4 + SAM
RDL B enzymes: BssHII and BseRI, NEB 4
26

To obtain A
12
,

A
24
and A
48,
pETb25b+ vectors containing A
6
, A
12
,

A
24
were subjected to
RDL A and B RE digestions respectively. Appropriate bands are excised from the
agarose gel, gel purified, ligated and then transformed into TOP10 cells. The end product
of this is an insert with double the number of the parent pentamers present.
For A
96
and

A
192
, following set of enzymes was used:
RDL A enzymes: AcuI, BglI and BssHII, NEB 4 + SAM
RDL B enzymes: BssHII and BseRI, NEB 4
The cloning procedure was the same as for A
12
,

A
24
and A
48
except the vectors
which were A48 and A96 respectively.

4.4 Recombinant Synthesis of SP1 into pcDNA3.1+ vector
The minigene containing the ER SS and N-glycosylation sequence (named as
SP1) was incorporated into in the pIDTSMART-KAN vector.
pcDNA3.1+ (Invitrogen
®
) and pIDTSMART-KAN vector (Integrated DNA
Technology) containing minigene were transformed into chemically competent TOP10
TM

cells are plated onto ampicillin and kanamycin resistant plates respectively. After an
overnight incubation at 37°C, the colonies were selected and inoculated into 4 ml TB
culture medium including 4 μl of 100mg/ml ampicillin and kanamycin respectively.
These were incubated overnight in a shaker incubator at 37°C and then purified to obtain
the plasmids using QIAprep Spin Miniprep Kit, QIAGEN.
27

Digestion: Minigene vector and pcDNA3.1+ were both digested using 10 units of NheI
and XhoI in the presence of NEB 2 and 10X BSA at 37
o
C for 3 hrs. The digested pcDNA
vector was then treated with CIP for 1 hr at 37
o
C. This was followed by heat deactivation
at 75
o
C for 10 mins.
Agarose Gel Electrophoresis: Digested minigene mixed with bromophenol blue loading
dye was loaded onto 1% Low Melting Point Agarose Gel (Bio-rad Bat# 161-3100). After
running the gel for 30 mins at 100V, the gel was analyzed using the Typhoon 8610
(variable mode imager). The minigene band was then excised from the gel and purified
using QIAquick Gel Extraction Kit, Qiagen. The CIP’ed pcDNA3.1+ vector was only
spin column purified using the same kit.
Ligation: Gel purified minigene and pcDNA3.1+ were ligated with 1:1 insert to vector
ratio respectively with the help of T4 DNA Ligase (InvitrogenTM) and ligase buffer. This
reaction mixture was then left at room temperature for 1 hr.
Transformation: The ligation mixture was then transformed into E. coli TOP10 cells
and plated onto ampicillin plates.
Confirmation of the insertion of the desired gene insert (i.e. minigene) into
pcDNA3.1+ vector: The colonies obtained were inoculated, stored as DMSO stocks and
purified. The sequence of the ER SS+N-glycosylation minigene (~200bp) was confirmed
from DNA sequencing result given by the Norris DNA Core Facility, USC. A primer
28

present upstream of the minigene in pcDNA3.1+ vector and T7 terminator primer
flanking the minigene sequence were used to get the sequence result.

4.5 Recombinant Synthesis of A96 and A192 into pcDNA3.1+ vector
DMSO stocks of A96 and A192 pentamers constructed in pETb25b+ vector and
pcDNA3.1+ vector were inoculated and purified with the QIAprep Spin Miniprep Kit,
Qiagen.
Digestion: The A96/192 pET25b+ vector was digested with 10 units of XbaI and XhoI in
presence of NEB 4 and 10X BSA at 37°C for 3 hrs. pcDNA3.1+ vector was digested with
10 units of NheI and XhoI using NEB 2 and 10X BSA at 37°C for 3 hrs. XbaI and NheI
produce same overhangs, thus, producing compatible or sticky ends.
Agarose gel electrophoresis: After running the digested vectors on the gel, bands of A96
and A192 (~1500 bp and ~3000bp) and pcDNA3.1+ vector (~5200bp) were cut and gel
purified.
Ligation: Inserts A96/192 were ligated with the purified pcDNA3.1+ vector using T4
DNA ligase and ligase buffer.
Transformation and screening of colonies: Part of the ligation mixture was then
transformed into TOP10
TM
cells and plated onto ampicillin plates. Colonies were selected
from the plates on the following day, diagnostic digested and sequence confirmed from
USC’s Norris DNA Core Facility.
29

4.6 Recombinant synthesis of SP3-A
192
into pcDNA3.1+ vector
SP1- A
192
was digested with BspEI in presence of NEB 3 at 37°C for 3hrs. After
gel purification, only the vector was self-ligated with the help of T4 DNA ligase and
ligase buffer. The self-ligated mixture was then transformed into TOP10 cells and plated
on ampicillin containing TB agar plates for overnight incubation at 37°C.

4.7 Protein Purification by ITC
For this purpose, a plasmid of SP1-A192 was transformed into BLR cells for
protein expression of the glycosylated ELP-Alanine. A starter culture of 50 ml+50 μl
ampicillin was inoculated and incubated overnight at 37°C in the shaker incubator. The
pellet of the overnight culture
The culture was centrifuged at 4000 rpm at 4°C for 10 mins and the pellet was
resuspended in cold PBS. The suspended cells were lysed using the sonicator.
Cold Spin: To eliminate the cell debris, the sonicated product was subjected to a cold
spin at 12000 rpm, 4°C for 15 mins so that the supernatant contained the soluble ELP.
The insoluble debris present in the pellet was discarded. The supernatant was mixed with
0.5% PEI and incubated on the ice for 10-20 mins. This caused the precipitation of DNA
and other cellular debris that was eliminated through centrifugation at 12000 rpm, 4°C
for 15 mins. The solution was warmed to 37°C in a water-bath and 3M sodium chloride
was added to lower the T
t
of the ELP. The pellet of the overnight culture was resuspended
in filtered PBS and used for re-inoculation of larger media volume. After overnight
30

incubation at 37°C, 250 rpm, this culture was centrifuged at 3000 rpm, 4°C for 10
minutes (Sorvall
®
RC 3C Plus). The pellet was resuspended in cold PBS and sonicated to
lyse the cells.
Cold Spin: Centrifugation of the sonicated product at 12000 rpm, 4 °C for 15 minutes
yielded supernatant with ELP and pelleted out the insoluble cellular debris that was
discarded. PEI (Aldrich Chemistry Cat#408700-1L) was mixed to a final concentration of
0.5% and placed on ice for 10-20 minutes. DNA and cellular debris was pelleted out by
centrifugation of this solution at 12000 rpm for 15 minutes at 4 °C. The supernatant was
placed in water-bath pre-warmed at 37°C water bath for 10 minutes along with the
addition of 3M of sodium chloride to reduce the T
t
of the ELP.
Hot Spin: The solution was centrifuged at 37°C for 10 minutes, 4000 rpm to pellet out
the ELP and discard debris present in the supernatant. The ELP containing pellet was
resuspended on ice in 15 ml PBS.
Cold Spin: The suspension was again centrifuged at 12000 rpm at 4°C for 10 minutes to
obtain purified ELP in the supernatant and eliminate the insoluble matter pellet.
To achieve high level of purity of ELP, at least two additional rounds of ITC were
undertaken.
31

4.8 Measurement of T
t
using Beckman Coulter DU 800 UV-vis spectrophotometer
The transition temperature is defined at the maximum first derivative of the
optical density at 350 nm. Transition temperatures of SP1-A
192
and ELP VPGSG-192
were characterized at 10, 25, 50 and 5, 10, 25, 50, and 100 μM ELP in phosphate buffer
respectively. The temperature was raised at 1°C/min on a Beckman DU800 UV-vis
spectrophotometer equipped with a multicell Peltier temperature controller [31].

4.9 SDS PAGE
To check the purity of the ELPs obtained from ITC, PAGEr
®
GOLD Precast 4-
20% gradient gels (LONZA) were used. 4X sodium dodecyl sulfate (SDS) loading buffer
containing glycerol and bromophenol blue was added to the protein samples. The
proteins were denatured at 95 °C for 5 minutes using heat block. 40 μg of the ELPs were
loaded into the wells. The ELPs were stained with copper chloride [41]. Kaleidoscope
protein ladder (Bio-rad laboratories) was used to determine the approximate molecular
weight of the protein. The Chemi-doc instrument (Bio-rad laboratories) was used to
analyze these gels.

4.10 Cell Culture and transfection method
Cell line and vector
 Transformed primary Human Embryonic Kidney cells 293 (American Type
Culture Collection, ATCC Number CRL-1573)
32

 Vectors used were Green Fluorescent Protein Expression, EGFP, SP1-A
192
, SP2-
A
192
and SP3-A
192
. The vector concentration was 100ng/uL with an approximate
260nm/280nm purity of 1.8.
Cell Culture, Transfection Reagents and media
 DOTAP:DOPE (1:1) liposome
 Polyethylenimine, Linear MW 25,000 (PEI), PolySciences, Inc. (Warrington,
Pennsylvania USA), Catalog No.23966
 Dulbecco’s Minimum Essential Medium (Cellgro, Cat. No. 10-013-CV)
 Trypsin EDTA 1X (Cellgro, Cat. No. 25-052-CI)
 Dulbecco's Phosphate-Buffered Saline, 10X with calcium and magnesium
(Cellgro Catalog Number: 20-030-CV)
 Fetal Bovine Serum, Regular (Catalog Number: 35-010-CV)

4.10.1 Starting HEK 293 Cell Cultures from Frozen Stocks
Growth Medium: DMEM (Dulbecco’s Minimum Essential Medium + 10% fetal bovine
serum).
1. HEK 293 cells were thawed rapidly by briefly immersing the vial in a 37
o
C water bath
(2-3 minutes with constant agitation). Upon thawing, the outside of the vial was wiped
with 70% EtOH and then transferred the contents of the vial to a T25 flask.
33

2. 9 ml of medium was then added to the flask followed by gentle swirling of the flask to
distribute cells evenly over the growth surface. The culture was incubated in a 37
o
C, 5%
CO2, humidified incubator.
3. The next day, the cells were examined under a microscope. Healthy cells displayed a
flat morphology and adhered well to the plate. The medium was replaced with fresh, pre-
warmed growth medium every 3 days.
4. Cell cultures were split every 3 days as per the need or when they reached 70–80%
confluency.

4.10.2 Passaging HEK 293 cells
The medium was aspirated and the cells were washed with pre-warmed sterile
PBS. The cells were then detached using 1–2 ml of trypsin-EDTA for 1–2 min. To stop
trypsinization, growth medium was added followed by resuspension of the cells gently
but thoroughly. Cells were counted using haemocytometer. Then desired number of
viable cells was transferred to a new culture flask or plate containing an appropriate
volume of growth medium with gentle rocking of the flask/plate for even distribution of
the cells.
34

4.10.3 Transfection of different plasmids into HEK 293 cells
All transfections were performed using DMEM without FBS.
4.10.3.A) GFP

and

EGFP

transfection into HEK 293 cells using Liposome
(DOTAP:DOPE::1:1)
 0.4*10
5
cells were seeded into a 24 well plate
 45-50% confluency before transfection
 Ratios of Liposome/DNA : 16:1, 8:1, 4:1, 2:1
 1 μg DNA, 0.5 ml medium
4.10.3.B) GFP

and

EGFP

transfection into HEK 293 cells using L-PEI
 0.5*10
5
cells were seeded into a 24 well plate
 50-55% confluency before transfection
 Ratios of N/P: 160:1, ,80:1, 60:1, 40:1, 20:1
 1 μg DNA, 0.5 ml medium
N/P = [1μl PEI stock solution * 23.2nM nitrogen residue] / [1 μg plasmid * 3nM
Phosphate residue]
35

4.10.3.C) GFP

transfection into HEK 293 cells using L-PEI
 0.5*10
5
cells were seeded into a 24 well plate
 40-45% confluency before transfection
 Ratios of N/P: 40:1
 0.4, 0.8, 1.6, 3.2 μg DNA, 0.5 ml medium
4.10.3.D) A
96
and

SP1-A
192
transfection into HEK 293 cells using L-PEI
 1.2*10
5
cells were seeded into a 6 well plate
 50% confluency before transfection
 2.5 μg DNA, 2.5 ml medium N/P ratio : 40:1
 72 hrs after transfection cells were lysed using lysis buffer
 Phase transition was tested using 3M salt (1ml medium taken from the plate)
4.10.3.E) GFP, SP1- A
192
, A
192
,

SP3- A
192
transfection into HEK 293 cells using L-PEI
 0.5*10
5
cells were seeded into a 6 well plate
 55-60% confluency before transfection
 Ratios of N/P: 40:1
 1 μg DNA, 2.5 ml medium
36

CHAPTER 5
RESULTS
5. 1 ELP VPGAG Library constructs
This gel picture shows the ELP library of VPGAG of lengths ranging from 6, 12,
24, 48, 96, 192 pentamers. A diagnostic digest was performed on pet25b+ vector
consisting of different ELP pentamers using XbaI and BamHI in presence of NEB 3 for
one hour at 37
o
C. The digests were then run on 1% agarose gel including SYBR Safe and
visualized using gel documentation system.

Figure 11: ELP VPGAG library

37

Table 3: Expected band size for respective length of ELP
ELP Length
(pentapeptides)
Expected Band
Size (base pairs)
6 90
12 180
24 360
48 720
96 1440
192 2880

38

Fig.12: SP1 sequence consisting of ER SS and 8 NGS glycosylation units is shown in
the block.

5.2 Transition Temperature for ELP A192 at different concentrations
ELP A
192
was purified using ITC protein purification technique and its T
t

was characterized as per the changes in concentrations using Beckman Coulter DU 800
UV-vis spectrophotometer. It was observed that as the concentration increases, the T
t

decreases. A
192
at 50 μM shows T
t
at approximately 32
o
C indicating that this length at a
little higher concentration could give phase transition temperature comparable to human
body temperature.
39

Figure 13: T
t
of A
192
at different concentrations

5.3 Determination of protein expression of A
96
and SP1- A
192
1ml of supernatant was collected from 6 well plates containing HEK 293 cells
transfected with different plasmid constructs and medium from lysed cells after 72 hrs of
transfection. 3M NaCl was added to the pre-warmed samples at 40
o
C to check for
turbidity (indicating ELP aggregate formation). SDS PAGE of these samples was
conducted to check for the presence of any glycosylated ELP or only ELP. No turbidity
or no bands were observed.

Figure 14: Determination of expression of glycosylated ELP or only ELP in HEK 293
cells using SDS PAGE (4.10.3.D)
40

Table 4: Samples introduced in each lane for SDS PAGEshown in Figure 14
Lane Sample
1, 6 Protein molecular weight marker
2 A
96
, Supernatant
3 GFP + A
96
, Supernatant
4 GFP + A
192
, Supernatant
5 GFP + SP1-A
192
, Supernatant
7 A
96
, Cell Lysis
8 GFP + A
96
, Cell Lysis
9 SP1-A
192
Cell Lysis
10 GFP + SP1-A
192
, Cell Lysis

5.4 Transfection of HEK 293 cells using different pcDNA3.1+ constructs
5.4.1 Liposome as transfection agent
Very low transfection efficiency with most of the dead cells fluorescing was
observed with liposomal transfection agent. The cells were sparsely adhered to the plate
with a low confluency level. Amongst the ratios tried for transfection, Liposome:EGFP =
4:1 showed TE of approximately 8% and nearly 5-6% for Liposome:GFP = 8:1, 16:1
ratios (Fig. 15).
41

Figure 15: FITC and DIC images of GFP and EGFP transfection into HEK 293 cells
using Liposome as transfection agent (4.10.3.A)

42

5.4.2 L-PEI as transfection agent [17]
Transfection of GFP vector using L-PEI with higher N/P ratios 160:1, 80:1, 60:1
and higher DNA concentrations of 1.6μg and 3.2μg proved toxic to HEK 293 cells
(Fig.16, 17).
Between 20-25% fluorescence was observed with N/P ratio of 40:1 with 0.4μg
and 0.8μg of GFP plasmid (Fig 17). However, less number of fluorescing cells was
observed with N/P ratio of 40:1 and 1μg GFP DNA (Fig.16). The variation could be due
to poor cell handling technique.
43

Figure 16: FITC and DIC images of GFP and EGFP transfection into HEK 293 cells
using L-PEI as transfection agent (4.10.3.B)

44

Figure 16: FITC and DIC images of GFP and EGFP transfection into HEK 293 cells
using L-PEI as transfection agent (4.10.3.B) continued

45

Figure 17: FITC and DIC images of GFP and EGFP transfection into HEK 293 cells
using L-PEI as transfection agent (4.10.3.C)

46

Figure 17: FITC and DIC images of GFP and EGFP transfection into HEK 293 cells
using L-PEI as transfection agent (4.10.3.C) continued

Transfection experiment was scaled up to a 6 well plate to increase the expression
of ELP protein as its T
t
is inversely proportional to concentration and for better handling
of HEK 293 cells. Expression of GFP vector alone and along with SP1-A
192
, SP2-A
192
,
SP3- A
192
evaluated in HEK 293 cells using L-PEI with a N/P = 40:1 resulted in
approximately 30% of fluorescing green cells. Under these conditions, it can be observed
from Fig.18 that the transfected cells showed a clear morphology in comparison to
previous experiments. The efficiency of GFP expression in co-transfection experiments is
approximately half of that observed in GFP alone transfection. This could be attributed to
half the amount of GFP used for co-transfection than that used for GFP alone.
47

Figure 18: FITC and DIC images of GFP and EGFP transfection into HEK 293 cells
using L-PEI as transfection agent (4.10.3.E)

48

CHAPTER 6
CONCLUSION
Elastin-like polypeptides with alanine as a guest residue in VPGXG pentapeptide
repeats of different lengths can be genetically engineered using Recursive Directional
Ligation Technique. Similarly, pcDNA3.1+ vector containing ERSS+NGS+ELP gene
was successfully constructed using recombinant technique.
ELP A
192
protein can be expressed in BLR cells and purified using ITC protein
purification method. T
t
of ELP A
192
present in pet25b+ vector was found to be around
31.7 C at 50 μM. It was seen that as the concentration decreased, the T
t
of A
192
increased.
Transfection of mammalian HEK293 cells with GFP and different constructs of
pcDNA3.1+ vectors using L-PEI transfection agent was optimized. It was found that
transfection efficiency between 20-25% could be achieved with N/P ratio of 40:1, 1μg
DNA in a 6 well plate. It was observed that cells in cluster were transfected with better
efficiency than cells that were sparsely distributed. Transfection of up to 1μg DNA of
different pcDNA3.1+ constructs containing ELP alone or ELP+N-glycosylation sequence
did not cause any obvious toxicity to HEK 293 cells.
49

Phase transition of SP1-A
192
, A96 in the lysed cells and medium collected after 72
hrs of transfection of HEK 293 cells was not observed. SDS PAGE of the same samples
did not show any protein bands. This indicates that either HEK 293 cells are not
expressing the glycosylated ELPs or the concentrations are too low to detect.
50

CHAPTER 7
FUTURE DIRECTIONS
The objective to determine the presence of N-glycosylated ELPs in mammalian
HEK293 cells could not be achieved. To resolve this, incorporation of a hexahistidine tag
into the pcDNA3.1+ constructs containing only ER SS+NGS units sequence, ER
SS+NGS+A
192
and A
192
could be done. This followed by western blot experiments using
appropriate primary and secondary antibodies against histidine tag would inform about
the expression and localization of the N-glycosylated ELP [36].
Presence of N-linked sugars can be determined by its cleavage using peptide N-
glycosidase F followed by a MALDI analysis [35]. This would help to analyze the
structures and types of sugar present. Further, it would indirectly indicate that the
endoplasmic reticulum signal chosen successfully directs the protein to the ER lumen for
post-translation modification (namely N-glycosylation in this case).
51

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Asset Metadata

Creator Parakh, Sejal (author)

Core Title Genetic engineering of thermally sensitive elastin-like polypeptide and its expression in HEK 293 cells

School School of Pharmacy

Degree Master of Science

Degree Program Pharmacy / Pharmaceutical Sciences

Publication Date 08/06/2010

Defense Date 06/04/2010

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

Tag elastin-like polypeptide,inverse transition cycling,N-glycosylation,OAI-PMH Harvest,recursive directional ligation

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Mackay, John Andrew (committee chair), Hamm-Alvarez, Sarah F. (committee member), Okamoto, Curtis Toshio (committee member)

Creator Email sejal.parakh@gmail.com,sparakh@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3329

Unique identifier UC1465591

Identifier etd-Parakh-3976 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-376384 (legacy record id),usctheses-m3329 (legacy record id)

Legacy Identifier etd-Parakh-3976.pdf

Dmrecord 376384

Document Type Thesis

Rights Parakh, Sejal

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu