University of Southern California Dissertations and Theses

Stable cell lines expressing elastin-like polypeptide fusions with epidermal growth factor receptor modulate gene expression in a heat dependent manner

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Stable cell lines expressing elastin-like polypeptide fusions with epidermal growth factor receptor modulate gene expression in a heat dependent manner

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Content
Stable cell lines expressing elastin-like polypeptide fusions with
epidermal growth factor receptor modulate gene expression
in a heat dependent manner

by
Geetha Boddu

A Thesis Presented to the
FACULTY OF THE USC SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the requirements for the Degree
MASTER OF SCIENCE
(Pharmaceutical Sciences)
August 2021

Copyright 2021 Geetha Boddu

ii
Acknowledgements

I would like to express my sincere gratitude to Dr. J. Andrew MacKay for his continuous
guidance and support of my research and my Ph.D. application over the past two years. I am
grateful for his patience, motivation, immense knowledge, and valuable discussions/suggestions
about my experiments. In addition, I would like to thank my committee members Dr. Curtis
Okamoto, Dr. Paul Seidler, for their support and insightful knowledge.

I would like to thank Hugo Avila for being my mentor and guiding me on the right path.
I’m grateful for all his suggestions, and I appreciate him for everything he taught me and his
integral and key role in my research. I would like to thank Anh Truong for all the help and support
he has done for me. I also want to thank our lab members Santosh Peddi, Hao Guo, Jingmei Yu,
Alvin Phan, Atham Ali, for all the help they have given me and all the good times we have had
together. Finally, I would like to thank my husband and son for their support throughout my
research.

iii
Table of Contents
Acknowledgements ____________________________________________________________ ii
LIST OF TABLES ____________________________________________________________ iv
LIST OF FIGURES ____________________________________________________________ v
Abstract ___________________________________________________________________ viii
Graphical Abstract: __________________________________________________________ ix
CHAPTER ONE: INTRODUCTION ______________________________________________ 1
CHAPTER TWO: MATERIALS AND METHODS _________________________________ 11
2.1. Cell culture and Stable cell generation _______________________________________ 11
2.2. Western blot ___________________________________________________________ 12
2.3. Live cell imaging _______________________________________________________ 12
2.4. Kinetic western blot assay ________________________________________________ 13
2.5. RT-qPCR analysis ______________________________________________________ 14
CHAPTER THREE: RESULTS _________________________________________________ 20
3.1. Stable cell line generation ________________________________________________ 20
3.2. BCA assay and Western blot ______________________________________________ 27
3.3. Live-cell imaging _______________________________________________________ 28
3.4. Kinetic western blot assay ________________________________________________ 31
3.5. RT-PCR analysis _______________________________________________________ 34
CHAPTER FOUR: DISCUSSION _______________________________________________ 48
CHAPTER FIVE: CONCLUSIONS ______________________________________________ 50
REFERENCES ______________________________________________________________ 51

iv
LIST OF TABLES

Table 1 List of various reagents used for transfection 11
Table 2 List of various reagents used to prepare 2X master mix 16
Table 3 Thermocycler conditions for reverse transcription reaction 16
Table 4 List of reagents for qPCR reaction 17
Table 5 Thermocycler conditions for qPCR reaction 17
Table 6 Dilutions to make 100uM stock 18
Table 7 List of primers used for qPCR by SYBR Green method 18
Table 8 List if reagents for qPCR reaction for TaqMan assay 18

Table 9 Thermocycler conditions for TaqMan assay 19
Table 10 BCA assay to detect EGFR-A96 and EGFR-V96 protein concentrations 27
Table 11 Standard graph for BCA assay 32
Table 12 BCA assay to know protein concentrations 32
Table 13 RNA purity was determined by using Thermo Scientific Nanodrop 2000
spectrophotometer 34
Table 14 EGFR TaqMan assay for EGFR-eGFP-V96 and EGFR-eGFP-A96 array
plates 37
Table 15 Fold change in gene expression of EGFR-eGFP-V96 cells compared to
EGFR-eGFP-V96 after thermal stimulation 38

v
LIST OF FIGURES
Figure 1 EGFR structure 2
Figure 2 Extracellular region of EGFR and ligand induced EGFR activation 3
Figure 3 EGFR signaling pathway 3
Figure 4 RAS-RAF-MEK-ERK/MAPK pathway 5
Figure 5 PI3K-AKT-mTOR pathway 6
Figure 6 JAK-STAT signaling pathway 7
Figure 7 PLC-PKC pathway 8
Figure 8 Steps involved in live-cell imaging 13
Figure 9 EGFR-eGFP-A96 stable cell line generation by FACS (1
st
round sorting) 22
Figure 10 EGFR-eGFP-A96 stable cell line generation by FACS (2
nd
round sorting) 23
Figure 11 EGFR-eGFP-A96 stable cell line generation by FACS (3
rd
round sorting) 24
Figure 12 EGFR-eGFP-A96 stable cell line generation by FACS (4
th
round sorting) 25
Figure 13 EGFR-eGFP-V96 cell sorting by FACS 26
Figure 14 Western blot results showed that generated stable cell lines are expressing
EGFR-eGFP-ELP proteins 27
Figure 15 Characterization of EGFR-eGFP-A96 cell line transition temperature 28
Figure 16 Characterization of EGFR-eGFP-V96 cell line transition temperature 29
Figure 17 EGFR-eGFP-V96 cells were evaluated for microdomain transition
temperature and temperature-sensitive variant exhibited
Tt at 31.8ºC 30
Figure 18 With temperature increase, around 30ºC, considerable proportion of
temperature-sensitive cells were transitioning while EGFR-eGFP-A96
vi
doesn’t 31
Figure 19 Temperature-dependent activation of p-ERK1/2 was observed in
the generated stable cell line 33
Figure 20 Fold change for EGFR-eGFP-A96 cells at various time points for
GAPDH, Fos, Jun, Myc 35
Figure 21 Fold change for EGFR-eGFP-V96 cells at various time points for
GAPDH, Jun, Myc 35
Figure 22 Higher gene expression at 60 min for EGFR-eGFP-V96 cell line 36
Figure 23: Expression levels of various upregulated genes such as RELB, RHOG,
PIK3C2A, RRAS, MUC1, NRAS, PIK3CA, MAPK10 41
Figure 24: Expression levels of various upregulated genes such as RHOC, AKT2,
CHUK, ARAF, MAPK9, SHC1, NKFB2, STAT3 41
Figure 25: Expression levels of various upregulated genes CSK, PRKCA, MAP2K2,
MAP2K1, MAP2K4, MAPK1, MAP2K7, RHOA 42
Figure 26: Expression levels of various upregulated genes such as PRKCG, CBL,
PRKCZ, MAPK3, IKBKG, SOS2, VAV1, HRAS 42
Figure 27: Expression levels of various upregulated genes such as PIK3CD,
ERBB2, NFKB1, PIK3R2, CAV2, CAV1, VAV3, DIRAS3 43
Figure 28: Expression levels of various upregulated genes such as GRB2, REL,
EPS8, STAT1, PXN 43
Figure 29: Expression levels of various upregulated genes such as RASA1,
CDH1, PAK1, IKBKB, AKT3, RAF1, PIK3CB 44

vii
Figure 30: Expression levels of various upregulated genes such as RAB5A,
PRKCD, SOS1, BRAF, JAK1, KRAS, CTNNB1 44
Figure 31: Expression levels of various upregulated genes such as RHOB,
ELK1, PLCG1, NCK1, RAC1, MAPK8, JAK2 45
Figure 32: Expression levels of various upregulated genes such as AKT1,
PRKCB, SRC, PTK2, IKBKE, PRKCE, MAP3K1 45
Figure 33: Expression levels of various upregulated genes such as RELA,
SHC3, EGF, RRAS2, GAB1, VAV2 46
Figure 34: Expression levels of various upregulated genes such as MRAS,
PDK1, EGFR, PIK3R1, PIK3C2B 46
Figure 33: Expression levels of various upregulated genes such as RND3, ABL1,
MYC, PRKCQ, JUN 47

viii
Abstract

Ligand-mediated EGFR forms homo or hetero dimers followed by autophosphorylation.
After autophosphorylation, internalization occurs, which results in the activation of EGFR cell
signaling. Previous researchers in the MacKay lab have designed ELP fusion proteins to modulate
Epidermal Growth Factor Receptor signaling. Dr. Mackay’s lab utilized the reversible phase
behavior of ELP fusions to regulate cell signals. For example, the substitution of the ligand EGF
by EGFR fused ELP can rapidly and transiently activate downstream signaling. Below its
transition temperature, EGFR-V96 remains soluble. In contrast, above its transition temperature,
EGFR-V96 fusions cluster, phosphorylate and internalize, initiate downstream kinase signaling.
As a negative temperature-independent control, EGFR-A96 was demonstrated to avoid clustering
at physiological temperature. Therefore, it has been selected as a control that does not activate
signaling proteins to modulate gene expression. Ligand EGF, not only activates the EGF receptor,
but it also activates other kinase receptors like HER2. Therefore, it was necessary to develop a tool
to understand EGF receptors alone explicitly. Towards this hypothesis, Zhe developed a switch to
activate EGFR alone in mammalian cells. This project seeks to understand whether EGFR fused
ELPs are temperature sensitive. What biological effects do they have? Can EGFR-ELP fusions
activates gene expression specific to EGFR signaling pathways? To answer these questions,
downstream targets of EGFR were measured by western blot and RT-PCR to quantify gene
expression of various EGFR signaling targets. This thesis concludes that EGFR-V96 specifically
activates ERK1/2; furthermore, this is associated with a dramatic enhancement in nuclear
transcription factors cFOS and JUN.

ix
Graphical Abstract:

1
CHAPTER ONE: INTRODUCTION

The epidermal growth factor receptor family of transmembrane receptor tyrosine kinases
(RTKs) includes four members such as EGFR, HER2, HER3, and HER4 [1]. EGFR is treated as
a prototype of all RTKs. EGFR is also known as HER-1 or c-erbB-1. EGFR plays a pivotal role in
cell signaling that governs the tumors. Overexpression of EGFR produces intense signals, which
activates a cascade of signals to progress uncontrolled cell division. EGFR is overexpressed in
many cancer types, and it is one of the targets for cancer therapy [2].

Human EGFR is located on chromosome 7 and codes for a 170kDa receptor tyrosine
kinase. It consists of an extracellular region, transcellular region, intracellular region, tyrosine
kinase domain, and a C-terminal regulatory region [3]. The extracellular region is further divided
into four domains, namely, domain I, domain II, domain III, and domain IV. Domain I and domain
III matches their sequence of about 37%, whereas domain II and domain IV are rich in cysteine
[4]. Domain I and III binds to EGF, whereas domain II plays a vital role in dimerization. Tyrosine
kinase domain comprises both N-lobe and C-lobe, which forms the ATP binding cleft [5]. The
carboxy-terminal region also includes tyrosine residues that auto phosphorylate upon ligand
binding [6].

2

Figure 1: EGFR structure [6]

EGFR primarily exists in two conformational states; tethered state extended/untethered
state. In the tethered state, the dimerization arm is hidden by four extracellular domains. In an
extended state, four domains stretch out in 130º arc to unmask the dimerization arm. Now, the
accessible dimerization arm permits coupling with another extended EGFR monomer, forming a
homodimer. However, this dimer unstable and may uncouple unless EGF is present. When EGF
binds to EGFR dimer, it becomes stabilized. After a second EGF binds, further changes within the
transmembrane and intracellular domains propagate signal for cell proliferation and survival [7].
3

Figure 2: Extracellular region of EGFR and ligand induced EGFR activation [7]

EGFR is activated by the binding of EGF agonists such as EGF, TGFα, amphiregulin (AR),
betacellulin (BTC), epigen (EPN), epiregulin (EPR), and heparin-binding EGF-like growth factor
(HB-EGF) [8]. EGF and transforming growth factor–α (TGF-α) are considered as the most
important ligands for EGFR downstream signaling activity. EGFR dimerizes upon activation by
an EGF agonist, which results in a transition from a monomer state to a dimer state. EGFR
dimerization results in the autophosphorylation of the tyrosine kinase domains [9,10]. Activation
of the receptor tyrosine kinase leads to the recruitment of several proteins at the receptors
intracellular portion. As a result, nuclear activation of genes related to cell proliferation,
differentiation, and cell growth occurs [11].

Figure 3: EGFR signaling pathway
130°
4
RAS-RAF-MEK-ERK/MAPK Pathway:
The Ras/Raf/MEK/ERK pathway plays a key role in the survival and development of
unchecked cell growth. Upon ligand binding, the activated EGFR binds to SHC (Src homology
and collagen). SHC becomes tyrosine phosphorylated after binding to EGFR. Then other adapter
protein GRB2 attach to tyrosine phosphorylated SHC as well as tyrosine-phosphorylated EGFR
members. GRB2 comprises one SH2 domain and two SH3 domains. These two SH3 domains
attach to proline-rich domains of SOS. Inactive RAS bind to GDP. SOS, a guanine nucleotide
exchange factor (GEF), catalyzes the interchange of GDP with GTP, which results in RAS protein
activation (“ON”) In contrast, GTPase activator proteins (GAPs) encourage GTP hydrolysis and
restore RAS to a GDP-bound state (“OFF”) [12]. Active Ras activates the kinase activity of RAF
kinase. In turn, activated RAF kinase activates MEK1 and MEK2. Activated MEK phosphorylates
ERK [13]. Then, the activated ERKs translocate to the nucleus and trigger various genes including
cFOS and JUN to promote growth, differentiation [14].

5

Figure 4: RAS-RAF-MEK-ERK/MAPK Pathway

6
PI3K-AKT-mTOR Pathway:

The PI3K-AKT-mTOR signaling pathway plays a pivotal role in cell proliferation and
survival. Upon ligand binding, the activated EGFR binds to the regulatory P85 subunit of PI3K.
Catalytic P110 domain of PI3K phosphorylates the three-hydroxyl group of the PIP2 to generate
PIP3. PIP3 stimulates AKT translocation to the plasma membrane. PDK1 phosphorylates
threonine 308 residue of AKT, whereas PDK2 and ILK phosphorylate serine 473 residues of AKT.
Now, active/phosphorylated AKT translocate from plasma membrane to cytosol. Akt
phosphorylates and suppresses/inactivates the TSC1/2 activity, leading to Rheb-mediated
activation of mTOR [15]. mTORC1 activates/phosphorylates P70S6K which phosphorylates
ribosomal protein S6 [16]. This results in protein synthesis, cell proliferation, cell survival.

Figure 5: PI3K-AKT-mTOR Pathway

7
JAK-STAT Pathway:

Upon binding of a cytokine or growth factor to its respective receptor, the JAK-STAT
pathway is activated. Upon binding, receptor-associated Janus Kinases (JAK) become activated
and phosphorylate each other. Jak-mediated phosphorylation activates STATs (Signal Transducers
and Activators of Transcription), and triggered STATs attach DNA to regulate gene expression
[17, 18]. In addition, when JAK is activated, it activates several downstream cascades, such as the
RAS-Raf-MEK-ERK pathway and PI3K-AKT signaling pathway.

Figure 6: JAK-STAT signaling pathway

8
PLC-PKC pathway:
Upon ligand binding, activated TKRs activate the phospholipase C (PLC) enzyme, which
cleaves PIP2 to generate second messengers, inositol 1,4,5-triphosphate (IP3), and Diacylglycerol
(DAG) [19]. Released IP3 acts on the endoplasmic reticulum’s IP3/Ca
+2
channel, thereby stimulates
the release of intracellular Ca+2 from the endoplasmic reticulum. DAG and released Ca
+2
activates
protein kinase C (PKC) [20]. Activated PKC enzymes are essential in several signal transduction
cascades.

Fig 7: PLC-PKC pathway

9
Elastin is one of the most significant extracellular matrix proteins found in tissues where
elasticity is required, such as large arteries, elastic ligaments, cartilage, lungs, and skin [21,22].
Elastin-like polypeptides (ELPs) are derived from tropoelastin, the precursor protein of elastin
[23]. ELPs are artificial, genetically encodable biopolymers with tunable thermo-sensitivity and
biocompatibility. ELPs are elastomeric proteins containing repeats of the Val-Pro-Gly-Xaa-Gly,
where Xaa represents guest amino acid residue. For example, If the guest amino acid residue is
alanine and the number of repeats are 96, referred to as A96 ELP, whereas If the guest amino acid
residue is valine and number of repeats are 72, referred to as V72 ELP. Typically, proline is
avoided at the guest amino acid residue since its presence can impede the ELP phase behavior.
The inclusion of tyrosine at the end of the ELPs promotes spectrophotometric analysis [24].

ELPs exhibit a quick and reversible phase transition behavior at a critical temperature. At
this temperature, ELPs undergo a conformational change from random coils to type II ß-turn
spirals known as transition temperature (Tt). ELPs are thermo-responsive polypeptides; below
transition temperature (Tt), they are highly water-soluble, whereas above transition temperature
(Tt), they self-assemble to form coacervates of different sizes [25]. This phase transition is
thermodynamically reversible. The Tt is finely tuned by altering ELPs chain length or guest amino
acid residue. Chain length or molecular weight of ELP increases by increasing the number of
repeats which results in lowering of Tt. If guest amino acid residue is hydrophobic like valine, the
transition temperature decreases. However, if it is hydrophilic amino acid guest residue like
alanine, the Tt is higher. In addition, as you increase the concentration of ELPs, it decreases the
transition temperature of ELPs.

10
ELP expression vectors are manufactured by recursive directional ligation (RDL) method.
ELPs are expressed in E. coli and purified by several rounds of inverse transition cycle (ITC). This
non-chromatographic method is rapid, economical, and coherent way of purifying ELP fusion
proteins. ITC utilizes thermally triggered phase separation behavior of ELPs to purify it [26,27].
ELP fusion protein’s phase transition temperature is triggered by adding salts such as NaCl (1-
3M) [28,29]. ELP fusion protein yield is increased by addition of stronger kosmotrops such as
ammonium sulfate, sodium citrate. Since samples become more concentrated subsequent rounds
of purification require lower salt concentrations.

Since ELPs are biocompatible, biodegradable, and non-immunogenic, this makes them
ideal for in vivo applications such as vehicle for drug delivery [30] and as intracellular switches.
As drug delivery systems, ELPs can prolong drug retention by modulating its hydrodynamic radius
yet maintaining its biological activity. As intracellular switches, ELPs can assemble, activate, and
deactivate various targeted cellular processes [31].

Previous researchers in the Mackay lab designed a protein switch that contributes to ON-
OFF regulation of EGFR, which entitles control of various downstream cascades. To design a
protein switch, they engineered chimeric receptors containing epidermal growth factor receptor
and ELPs and demonstrated their tunable modulation of downstream signaling through EGFR
clustering. This chapter supports the hypothesis that EGFR fused ELPs promotes temperature
triggered receptor internalization and transactivation of various transcription factors, leading to
change in gene expression to promote growth and differentiation.
11
CHAPTER TWO: MATERIALS AND METHODS

2.1. Cell culture and Stable cell generation

HEK293T cells were cultured in DMEM containing 10% FBS in a 37C incubator with 5%
CO2 supply. HEK293T cells were transfected with PcDNA3.1 EGFR-eGFP-A96 by using
Lipofectamine 3000 reagent according to Thermo Fischer Scientific protocol (#L3000015).
Transfection: Seeded HEK293T cells to be 70-90% confluent at transfection. Diluted
Lipofectamine™ 3000 Reagent (37.5uL) in Opti-MEM™ Medium (837.5uL) and mixed well.
Prepared master mix of DNA (52.2uL) by diluting DNA in OptiMEM™ Medium (797.8uL), then
added P3000™ Reagent (25uL) and mixed well. Added Diluted DNA to diluted Lipofectamine™
3000 Reagent (1:1 ratio). Incubated for 10-15 min at room temperature. Added DNA-lipid
complex to HEK 293T cells. Incubated cells for 4 days and checked fluorescence under
Epifluorescence microscope.

Table 1: List of various reagents used for transfection
When transfected cells reached 70-80% confluence in T-175 flask, aspirated the media and
washed cells with PBS. Trypsinized cells for 5 minutes and deactivated trypsin by adding DMEM.
12
Spun down the cells and cell pellet were resuspended in 1000uL DMEM (-Phenol Red) + 10%
FBS+Penstrep. Dispersed cell suspension passed through cell strainer (#352235, Bd Biosciences)
to remove cell aggregates and performed Fluorescence-activated cell sorting (FACS) to separate
GFP positive cells. GFP positive cells were collected in 750uL DMEM+10% FBS. To make sure
all the cells are GFP positive, 4 rounds of colony selection was performed. 3 rounds of colony
selection were performed by Hugo Avila. I performed the last round for EGFR-eGFP-A96 and
EGFR-eGFP-V96 cell line.

2.2. Western blot

The whole-cell lysate was used for western blot analysis. 25ug of EGFR-eGFP-V96,
EGFR-eGFP-A96, HEK293T proteins were loaded in each well of precast gel. HEK293T proteins
were used as a negative control. Initially ran the gel with low voltage (50V) for separating gel for
10 minutes, then used higher voltage (250V) for stacking gel. The gel was run until the dye reached
the bottom of the gel. Gel proteins were transferred to the nitrocellulose membrane using the iBlot2
dry blotting system (ThermoFisher Scientific, Waltham, MA). The membrane was blocked with
5% BSA for 1hour and incubated the membrane with anti-EGFR (#4267S, Cell Signaling
Technology, Danvers, MA), anti-GAPDH (#2118S, Cell Signaling Technology, Danvers, MA)
primary antibodies (1:1000) overnight at 4C on a shaker, subsequent incubation with anti-rabbit
secondary antibodies (1:2000) for 1hr. Proteins were visualized using Suprasignal
TM
West Dura
Extended duration substrate, and signal intensity was measured using the iBright system.

2.3. Live cell imaging

EGFR-eGFP-A96, EGFR-eGFP-V96 cells were cultured on 35mm glass bottom dishes
(MatTek Corporation, Ashland, MA) coated with Poly-D-Lysine (P7280, Sigma-Aldrich, St.
13
Louis, MO) and serum starved overnight before imaging. Experiments were performed in
triplicates. Confocal imaging was performed by Hugo Avila.

Figure8: Steps to Live-cell Imaging

2.4. Kinetic western blot assay

Seeded EGFR-eGFP-A96 and EGFR-eGFP-V96 cells in 6-well plates. When cells reached
70-80% confluence, cells were starved for 24hrs before temperature stimulation. EGFR-eGFP-
A96 and EGFR-eGFP-V96 cells were washed with cold PBS and cultured in cold DMEM with no
FBS. Cells were incubated for 1 hour at 4C. Next, they were washed cells with PBS (37C) and
added DMEM with no FBS (37C). EGFR-eGFP-A96 and EGFR-eGFP-V96 cells were lysed at
various time points such as 0, 10, 20, 40, 80, 160 min. EGFR-eGFP-A96 cells were treated with
1ng/mL of EGF and lysed at various time points such as 0, 10, 20, 40, 80, 160 min. A BCA assay
14
was performed to estimate unknown protein concentrations. 30 ug of EGFR-eGFP-A96, EGFR-
eGFP-V96 and EGF treated EGFR-eGFP-A96 proteins were loaded in each well of precast gel.
EGFR-eGFP-A96, EGF treated EGFR-eGFP-A96 cells were used as a negative control and
positive control respectively. Initially the gel was run with low voltage (50V) for 10 minutes,
before running at higher voltage (250V) for stacking. The gel was run until the dye reached the
bottom of the gel. Gel proteins were transferred to the nitrocellulose membrane using the iBlot2
dry blotting system (ThermoFisher Scientific, Waltham, MA). The membrane was blocked with
5% BSA for 1hour and incubated the membrane with anti-p-ERK1/2 (#4370S, Cell Signaling
Technology, Danvers, MA), anti-GAPDH (#2118S, Cell Signaling Technology, Danvers, MA)
primary antibodies (1:1000) overnight at 4C on a shaker, subsequent incubation with anti-rabbit
(Alexaflour 647, #A12463, Invitrogen by ThermoFisher Scientific) secondary antibodies (1:1000)
overnight at 4C. Signal intensity was measured using the iBright system.
2.5. RT-qPCR analysis

mRNA was extracted using PureLink
TM
RNA Mini Kit (#12183018A, Invitrogen by
Thermo Fischer Scientific) as stated in Invitrogen manufacturer’s protocol. cDNA was synthesized
using High-capacity RNA to cDNA reverse transcription kit (#4368814, Thermo Fisher
Scientific). The generated cDNA samples were diluted and used as a template for qPCR.

15
RNA extraction:

Protocol for Reverse Transcription (cDNA synthesis):
Workflow:

2X master mix preparation: Prepared 2X RT master mix by adding 10X RT buffer, 25X
dNTPmix, 10X random primers, reverse transcriptase, and nuclease free water. Mixed gently and
placed it on ice.

16
Component Volume/Reaction (uL)
10X RT buffer 2.0
25X dNTPmix 0.8
10X random primers 2.0
Reverse transcriptase 1.0
Nuclease free water 4.2
Total per reaction 10

Table 2: List of various reagents used to prepare 2X master mix
cDNA reverse transcription reactions preparation:
Added 10uL of RNA sample to 10uL of 2X RT master mix tube. Centrifuged tube to spin
down contents.
Note: Use up to 2ug of total RNA per 20uL reaction for best results.
To perform reverse transcription, below conditions were programmed in thermocycler.
Adjusted reaction volume to 20uL in thermocycler. Loaded the reactions into the thermocycler
and performed reverse transcription.
Step 1 Step 2 Step 3 Step 4
Temperature (°C) 25 37 85 4
Time 10 min 120 min 5 min ¥

Table 3: Thermocycler conditions for reverse transcription reaction
Protocol for qPCR:

Thawed 2XqPCR Master Mix (High ROX) (#QP03-01, Bioland Scientific LLC) and EGFR-
eGFP-V96 30min, 60min RNA samples on ice. 2XqPCR Master Mix (High ROX) is SYBR Green
I-based qPCR reagent. It consists of PowerPCR™ Hot Start Taq DNA polymerase, PCR buffer,
dNTPs, SYBR Green I fluorescent dyes, Mg2+ and ROX calibration dye.

17
1. Preparation of reaction solution: Added all the reagent solutions in a PCR tube on ice.
Component of sample Volume Final concentration
2XqPCR Master Mix with ROX) 25uL 1X
Forward primer (10uM) 1uL 0.2uM
Reverse primer (10uM) 1uL 0.2uM
Template DNA

Variable 10pg-1ug
Nuclease free water To 50uL

Table 4: List of reagents for qPCR reaction

2. Performed PCR using the following thermal cycling conditions.

Initial Denaturation 95C 10min
35-45 cycles 95C
60C
15sec
60sec
Melting curve analysis

Table 5: Thermocycler conditions for qPCR reaction
Preparation of stock PCR primers:
Before opening the lyophilized primer, it was spun down to ensure that the primer pellet is
at the bottom of the tube. The 100 µM primer stock solution was prepared by adding TE (10 mM
Tris pH 8, 1 mM EDTA) to primer pellet tube. This tube was used to make working primer
solutions as required and stored at -20°C for future usage. To prepare primers for use, this stock
was diluted 1:10, to give a concentration of 10 uM.

18
Primers TE (uL) to make 100uM stock
Fos-F 857
Fos-R 1155
Jun-F 1322
Jun-R 963
Myc-F 874
Myc-R 900
GAPDH-F 956
GAPDH-R 944

Table 6: Dilutions to make 100uM stock

Primers Sequence Tm (°C) GC content (%)
Fos-F 5’-GTG GAA CAG GAG ACA GAC CAA-3’ 56.7 52.4
Fos-R 5’-TCC TTC AGC AGG TTG GCA AT-3’ 57.2 50
Jun-F 5’-GAG CTG GAG CGC CTG ATA AT-3’ 57.2 55
Jun-R 5’-CCC TCC TGC TCA TCT GTC AC-3’ 57.4 60
Myc-F 5’-TTT CGG GTA GTG GAA AAC CA-3’ 54.1 45
Myc-R 5’-CAC CGA GTC GTA GTC GAG GT-3’ 58.3 60
GAPDH-F 5’-AGC CAC ATC GCT CAG ACA C-3’ 57.6 57.9
GAPDH-R 5’-GCC CAA TAC GAC CAA ATC C-3’ 53.8 52.6

Table 7: List of primers used for qPCR by SYBR Green method
Protocol for qPCR:
Thawed the TaqMan
TM
Fast Advanced Master Mix (# 4444557, Thermo Fischer Scientific)
and RNA samples on ice.
The following components were combined for the number of reactions required, plus 10% overage.
Components 96-well plate (Fast)
cDNA template + Nuclease free water 5uL
TaqMan Fast Advanced Master Mix (2X) 5uL
Total volume per reaction 10uL

Table 8: List if reagents for qPCR reaction for TaqMan assay

19
The PCR reaction was vortexed briefly to mix and centrifuged to bring the reaction mix to the
bottom of the tube as well as to eliminate air bubbles. The PCR reaction mix was transferred (10uL)
to each well of TaqMan array 96-well fast plate (#4418906, Applied Biosystems). The plate was
sealed with optical adhesive film and, centrifuged to bring the PCR reaction mix to the bottom of
the well and eliminate the air bubbles.
A plate document or experiment file was setup using the following conditions in
QuantStudio
TM
12K Flex Real-Time PCR system.
Real-Time PCR system UNG incubation Polymerase activation PCR (40 cycles)
Hold 50°C Hold 95°C Denature 95°C Anneal/ extend 60°C
QuantStudio
TM
12K Flex
Real-Time PCR system.
2 minutes 2 minutes 1 second 20 seconds

Table 9: Thermocycler conditions for TaqMan assay
TaqMan Reagents were selected to detect the target sequence in the fast run mode. Sample
volumes were entered as 10uL. The plate document or experiment file was opened that corresponds
to the reaction plate in the system software. The reaction plate was loaded to start the run.

20
CHAPTER THREE: RESULTS

3.1. Stable cell line generation

Stable cell lines exhibit long term protein expression, are more reproducible. Since this
study requires measuring downstream signaling of EGFR using qPCR, sustained gene expression
across all the cells is required to detect signals over time. Therefore, generation of stable cell lines
is a viable option to get consistent results as it eliminates the variation associated with repeated
transient transfection, which only transfects a minority subset of the cells.

There are different methods to generate stable cell lines such as generation of stable cell
lines using lentivirus and generation of stable cell lines by electroporation. To generate stable cell
lines by these two methods requires a kill curve to determine the minimal antibiotic concentration
required to kill untransformed cells. It is crucial to know the characteristics of the cell line we want
to transfect to be sure that the selection of antibiotics will work on our cell line. For instance, for
CHO cells, the selection antibiotic is G418 disulfate. However, HEK293T cells are resistant to
G418 disulfate due to the presence of neomycin resistance genes. Therefore, G418 disulfate is not
recommended for HEK293T cells. Since HEK293T cells were selected for this project, we chose
FACS (Fluorescence Activated Cell Sorting) to generate stable cell lines.

Flow cytometry measures the properties of cells while in a fluid stream. In FACS,
hydrodynamic focusing pushes through the laser, it refracts and scatters light at all angles. Forward
scattered (FSC) light is the amount of light scattered in the forward direction. The intensity of
forward scattering is directly proportional to cell size. Side scatter (SSC) is the light scattered at
all angles. The intensity of side scattering is directly proportional to the granularity of the cells.
21
Filtered scattered light and fluorescence is collected and converted to digitalized values that are
sorted in a file, which can be read by a software.

Figure 9(A), FSC-A Vs. SSC-A dot plots were used to identify my cell population of
interest and exclude dead cells and debris. Debris and dead cells tend to have a lower scatter level.
Therefore, they often are found at the bottom left corner of the FSC Vs. SSC dot plot. Each dot on
the graph represents a cell.

When cells pass through the laser, voltage pulses are generated. A processed voltage pulse
is determined by its area (A), height (H), and width (W). Width is interpreted as the time taken for
a cell to pass through the laser beam and represents the cell size, whereas height is the intensity of
the signal. There are different ways of discriminating doublets, such as ‘A’ vs ‘H’ (or) ‘W’ vs ‘H’,
shown in figure9 (B). These dot plots were used to remove the doublets and cell clusters. Cells
near the diagonal are singlets, while those off it are doublets and clusters. Cells off this diagonal
were excluded from the data.

In figure 9(C), GFP-positive cells were sorted from GFP-negative cells. Cell sorting was
repeated several times through subculturing, and the data was shown in figures 10, 11, 12. Our
previous lab researcher, Zhe Li, developed a stable cell line with a temperature-sensitive variant.
To make sure all cells were fluorescent, one round of cell sorting was performed with the EGFR-
eGFP-V96 cell line, and the data was shown in figure 13.

22

(A) FSC-A Vs SSC-A dot plot (B) Doublet exclusion dot plot

(C) FSC-A Vs FITC-A dot plot

Figure9: EGFR-eGFP-A96 cell lines generation by FACS (1
st
round sorting)
23

Figure: (A) FSC-A Vs SSC-A dot plot (B) Doublet exclusion dot plot

(C) FSC-A Vs FITC-A dot plot

Figure10: EGFR-eGFP-A96 stable cell lines generation by FACS (2
nd
round sorting)

24

Figure: (A) FSC-A Vs SSC-A dot plot

(B) FSC-A Vs FITC-A dot plot

Figure11: EGFR-eGFP-A96 stable cell lines generation by FACS (3
rd
round sorting)

25

Figure: (A) FSC-A Vs SSC-A dot plot (B) Doublet exclusion dot plot

(C) FSC-A Vs FITC-A dot plot

Figure12: EGFR-eGFP-A96 stable cell lines generation by FACS (4
th
round sorting)

26

Figure: (A) FSC-A Vs SSC-A dot plot (B) Doublet exclusion dot plot

(C) FSC-A Vs FITC-A dot plot

Figure13: EGFR-eGFP-V96 cell sorting by FACS

27
3.2. BCA assay and Western blot

BCA assay was performed to determine protein concentrations in whole cell lysate sample.

Abs1 Abs2 Abs3 Avg Conc.
(ug/mL)
Corrctd
conc.
(ug/mL)
Corrctd
conc.
(ug/mL)
Vol.
(uL)
d.H2o
(uL)
Total
Vol.
(uL)
A 3.165 3.177 3.18 3.174 2000
B 2.987 3.051 2.829 2.955 1500
C 1.795 1.811 1.811 1.805 1000
D 1.775 1.762 1.62 1.719 750
E 1.09 1.057 1.082 1.076 500
F 0.648 0.662 0.673 0.661 250
G 0.388 0.385 0.383 0.385 125
H 0.161 0.159 0.16 0.160 25
I 0.106 0.108 0.109 0.107 0
V96 0.72 0.783 0.793 0.765 336 1680 1.680 15.0 0.0 15
A96 0.916 0.942 0.931 0.920 438 2193 2.193 11.5 3.5 15

Table 10: BCA assay to detect EGFR-A96 and EGFR-V96 protein concentrations

Western blot was used to confirm EGFR-eGFP-ELP proteins. Whole cell lysate was used
for western blot analysis. Membrane was incubated with anti-EGFR and anti-GAPDH and signal
intensity was measured using the iBright system.

Figure14: Western blot results showed that generated stable cell lines are expressing
EGFR-eGFP-ELP proteins

Protein Expected M.W. (kDa)
EGFR 172
eGFP 27
ELP 40
EGFR-eGFP-ELP 240
GAPDH 36
GAPDH
EGFR-eGFP-ELP
EGFR-eGFP-A96
EGFR-eGFP-V96
28
3.3. Live-cell imaging

Stably transfected EGFR-eGFP-A96 live cells were subjected to a temperature ramp to
detect temperature triggered EGFR fusion clusters. Images were acquired with a confocal
microscope. Since EGFR-eGFP-A96 cells are temperature insensitive, they didn’t form
microdomains at the temperatures evaluated. To sum up, no EGF receptor fusion clusters were
detected at any physiological temperature.

Figure15: Characterization of EGFR-eGFP-A96 cell line transition temperature
29
However, EGFR-eGFP-V96 cells exhibited the confirmation of receptor internalization at
31.8°C. Formation of microdomains were determined by eye. Below transition temperature,
EGFR-eGFP-V96 ELPs were soluble, and as the temperature increases above the transition
temperature, EGFR-eGFP-V96 ELPs formed microdomains. In conclusion, EGFR-eGFP-V96
transitions at ~31.8°C, while EGFR-eGFP-A96 does not.

Figure16: Characterization of EGFR-eGFP-V96 cell line transition temperature

Soluble Coacervate
30
Temperature ramps of different cells were taken three times to triplicate the data. In the
first run, second run, and third run, 21 cells, 10 cells, and 29 cells were taken, respectively. To
characterize Tt, we looked at three different runs and created an average of three runs. Figure 17
was representing that cell's transition at 31.8C.

Figure17: EGFR-eGFP-V96 cells were evaluated for microdomain transition temperature
and temperature sensitive variant exhibited Tt at 31.8C (Image credit: Hugo Avila)

31
When we look at the proportion of the cells, as we increase the transition temperature, we
observed apparent differences in cells transitioning temperature for EGFR-eGFP-A96 and EGFR-
eGFP-V96. We noticed transitioning of the cells at 31C for temperature-sensitive variant.
However, for the EGFR-eGFP-A96, few cells displayed any signs of receptor internalization at
any given temperature.

Figure18: With temperature increase, above 30ºC a considerable proportion of temperature-
sensitive cells internalized EGFR-eGFP-V96, while EGFR-eGFP-A96 did not (Image credit:
Hugo Avila)

3.4. Kinetic western blot assay

From western blot and live-cell imaging data, it was confirmed that generated stable cell
lines expressing EGFR-eGFP-ELPs and characterized their transition temperatures. Next, kinetic
western blot assays were performed to see EGFR activation and downstream signaling in a heat-
dependent manner. To determine temperature triggered phosphorylation state of ERK1/2, EGFR-
eGFP-A96 and EGFR-eGFP-V96 cells were lysed after applying heat or EGF at various time
32
points such as 0, 10, 20, 40, 80 min. The BCA assay was performed to estimate unknown protein
concentrations. The membrane was incubated with anti-p-ERK and anti-GAPDH, and signal
intensity was measured using the iBright system.

Conc. (ug/mL) Abs
2000 3.09
1500 2.36
1000 1.76
750 1.41
500 0.99
250 0.59
125 0.35
25 0.14
0 0.1

Table 11: Standard graph for BCA assay
Time Sample Average Adjusted Conc.(ug/mL) Conc.(ug/uL) Vol
(uL)
H20
(uL)
Total(uL)
0min EGFR-A96 0.67 3.35 2111.13 2.1 14.0 1.0 15
EGFRA96+EGF 0.63 3.15 1977.80 2.0 14.9 0.1 15
EGFR-V96 0.9 4.5 2877.80 2.9 10.3 4.7 15
10min EGFR-A96 0.95 4.75 3044.47 3.0 9.7 5.3 15
EGFRA96+EGF 0.81 4.05 2577.80 2.6 11.4 3.6 15
EGFR-V96 1.08 5.4 3477.80 3.5 8.5 6.5 15
20min EGFR-A96 0.95 4.75 3044.47 3.0 9.7 5.3 15
EGFRA96+EGF 0.91 4.55 2911.13 2.9 10.1 4.9 15
EGFR-V96 0.99 4.95 3177.80 3.2 9.3 5.7 15
40min EGFR-A96 0.88 4.4 2811.13 2.8 10.5 4.5 15
EGFRA96+EGF 0.77 3.85 2444.47 2.4 12.1 2.9 15
EGFR-V96 1.06 5.3 3411.13 3.4 8.6 6.4 15
80min EGFR-A96 0.84 4.2 2677.80 2.7 11 4 15
EGFRA96+EGF 0.96 4.8 3077.80 3.1 9.6 5.4 15
EGFR-V96 1.13 5.65 3644.47 3.6 8.1 6.9 15
EGFR-A96 0.96 4.8 3077.80 3.1 9.6 5.4 15
EGFRA96+EGF 1.03 5.15 3311.13 3.3 8.9 6.1 15
EGFR-V96 1.26 6.3 4077.80 4.1 7.2 7.8 15

Table 12: BCA assay to know protein concentrations

y = 0.0015x + 0.1833
R² = 0.9946
0
0.5
1
1.5
2
2.5
3
3.5
0 500 1000 1500 2000 2500
Conc Vs Abs
33
In Figure 19, p-ERK1/2 protein levels were compared in temperature triggered and ligand
triggered responses overtime after applying heat or EGF. For positive control, EGFR-eGFP-A96
cells were treated with 1ng/mL of EGF as it suggests the physiological levels of EGF ligands in
humans. Both temperature triggered and ligand triggered responses resulted in rapid activation of
ERK1/2. However, p-ERK1/2 activation was transient. So, this is consistent with previously
published Zhe Li’s results. In addition, the phosphorylation state of ERK1/2 is longer with ligand-
mediated activation compared to temperature-triggered activation. In a nutshell, EGFR-eGFP-V96
enables transient activation of canonical ERK signaling in a temperature-dependent manner. Since
EGFR-eGFP-A96 is temperature insensitive, we don’t see the activation of ERK1/2.

0min 10min 20min 40min 80min

Figure19: Temperature-dependent activation of phospho ERK1/2 was observed in the
generated stable cell lines

p-ERK 1/2
GAPDH
p-ERK 1/2
GAPDH
p-ERK 1/2
GAPDH
EGFR-eGFP-V96
EGF treated EGFR-eGFP-A96
EGFR-eGFP-A96
34
3.5. RT-PCR analysis
The kinetic western blot assay confirmed that EGFR-eGFP-V96 can activate the ERK1/2
pathway in a heat-dependent manner. Once EGFR is activated, it activates several
genes/transcription factors. Some of these include c-Fos, c-Jun, c-Myc. These were measured as
various downstream targets by qPCR.
To analyze gene expression, mRNA was collected from EGFR-eGFP-A96, EGFR-eGFP-
V96 cell lines at 0, 30, 60, 120 min. The Purity of mRNA was determined by using Thermo
Scientific Nanodrop 2000 Spectrophotometer. A 260/280 ratio of ~2 indicates pure RNA. In a two-
step RT-PCR the first step includes Reverse Transcription (RT) of RNA by reverse transcriptase.
This reaction makes first-strand complementary DNA (cDNA). In the second step, cDNA is
amplified by PCR and measured.

RNA Sample Conc.(ng/uL) A260 (Abs) A280 (Abs) 260/280
EGFR-eGFP-A96 0min 749.4 18.74 8.99 2.08
EGFR-eGFP-A96 30min 151.5 3.79 1.82 2.09
EGFR-eGFP-A96 60min 422.3 10.56 5.12 2.06
EGFR-eGFP-A96 120min 142.8 3.57 1.69 2.11
EGFR-eGFP-V96 0min 589.0 14.73 7.21 2.04
EGFR-eGFP-V96 30min 539.2 13.5 6.7 2.0
EGFR-eGFP-V96 60min 389.5 9.7 4.7 2.1
EGFR-eGFP-V96 120min 202.0 5.05 2.42 2.09

Table 13: RNA purity was determined by using Thermo Scientific Nanodrop 2000
Spectrophotometer

To estimate best mRNA extraction time point, EGFR-eGFP-A96 and EGFR-eGFP-V96
cells were extracted at 0, 30, 60, 120min. As expected, EGFR-eGFP-A96 did not activate the ERK
pathway as well as we didn’t see the activation of the Fos, Jun, Myc genes. In contrast, fold change
is greater for Fos, Myc, Jun when EGFR-eGFP-V96 cells are extracted at 1 hour after thermal
stimulation.
35

Figure 20: Fold change for EGFR-eGFP-A96 cells at various time points for GAPDH, Fos,
Jun, Myc

Figure 21: Fold change for EGFR-eGFP-V96 cells at various time points for GAPDH, Jun,
Myc

0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
GAPDH FOS JUN MYC
Fold change
Target gene
EGFR-eGFP-A96 relative gene expression
at 0min at 30min at 60min at 120min
0
2
4
6
8
10
12
14
16
GAPDH JUN MYC
Fold change
Target gene
EGFR-eGFP-V96 relative gene expression
at 0min at 30min at 60min at 120min
36

Figure 22: Higher gene expression at 60 min for EGFR-eGFP-V96 cell line

From figure 19, it was evident that EGFR-eGFP-A96 serves as a negative control, and from
figure 20 and 21, it was apparent that the best time point to extract mRNA to get higher gene
expression is at 1 hour after thermal stimulation. Based on the above conclusions, EGFR-eGFP-
A96 and EGFR-eGFP-V96 lysed at 60min were taken for TaqMan assay to analyze various EGFR
genes.
Livak method or 2^
-DDCt
was used to calculate relative changes in gene expression from RT-PCR.
Calculations:

Step1: Normalize Ct of the target gene to Ct of the reference gene

DCt = Ct (target gene) – Ct (reference gene)

Step2: Normalize DCt of the test (EGFR-V96) to the DCt of the control (EGFR-A96)

DDCt = DCt (EGFR-V96) - DCt (EGFR-A96)

Step3: Calculate the fold difference in expression

2^
-DDCt
= Normalized expression ratio
0
200
400
600
800
1000
1200
1400
at 0min at 30min at 60min at 120min
Fold change
FOS gene
EGFR-eGFP-V96 relative gene expression
37

Raw CT Data Livak 18S
Gene A96 V96 ΔCt (A96) ΔCt (V96) ΔΔCt 2^-ΔΔCt
18S 21.132 22.456 NA NA NA NA
GAPDH 18.818 19.681 NA NA NA NA
HPRT1 22.944 23.812 NA NA NA NA
GUSB 24.765 24.316 NA NA NA NA
ABI1 35.001 34.151 13.869 11.695 -2.174 4.51272191
AKT1 24.359 24.520 3.227 2.064 -1.163 2.23922298
AKT2 25.471 26.351 4.339 3.895 -0.444 1.36037108
AKT3 24.094 24.398 2.962 1.942 -1.020 2.02791692
ARAF 25.024 25.874 3.892 3.418 -0.474 1.38895419
BRAF 26.056 26.303 4.924 3.847 -1.077 2.10964414
CAV1 27.744 28.186 6.612 5.730 -0.882 1.84292589
CAV2 26.068 26.515 4.936 4.059 -0.877 1.83655335
CBL 24.199 24.770 3.067 2.314 -0.753 1.68529173
CDH1 28.445 28.778 7.313 6.322 -0.991 1.98756111
CHUK 25.037 25.899 3.905 3.443 -0.462 1.37744994
CSK 28.237 28.927 7.105 6.471 -0.634 1.55186049
CTNNB1 20.553 20.769 -0.579 -1.687 -1.108 2.15546599
DIRAS3 36.901 37.308 15.769 14.852 -0.917 1.88818709
EGF 37.139 36.996 16.007 14.540 -1.467 2.76446625
EGFR 22.415 21.968 1.283 -0.488 -1.771 3.41290417
ELK1 24.115 24.321 2.983 1.865 -1.118 2.17045878
EPS8 25.223 25.568 4.091 3.112 -0.979 1.97109619
ERBB2 29.615 30.098 8.483 7.642 -0.841 1.79129084
FOS 32.043 22.758 10.911 0.302 -10.609 1,561.8056
GAB1 26.392 26.193 5.260 3.737 -1.523 2.87387798
GRB2 25.385 25.770 4.253 3.314 -0.939 1.91719778
HRAS 25.393 25.938 4.261 3.482 -0.779 1.71594026
IKBKB 27.532 27.838 6.400 5.382 -1.018 2.02510912
IKBKE 32.143 32.092 11.011 9.636 -1.375 2.59368253
IKBKG 28.471 29.030 7.339 6.574 -0.765 1.69936927
JAK1 24.233 24.472 3.101 2.016 -1.085 2.12137413
JAK2 29.048 29.233 7.916 6.777 -1.139 2.20228310
JUN 24.008 21.107 2.876 -1.349 -4.225 18.70041619
KRAS 28.424 28.661 7.292 6.205 -1.087 2.12431821
MAP2K1 26.156 26.808 5.024 4.352 -0.672 1.59327906
MAP2K2 25.937 26.612 4.805 4.156 -0.649 1.56808109
MAP2K4 26.827 27.474 5.695 5.018 -0.677 1.59880959
38
MAP2K7 28.170 28.798 7.038 6.342 -0.696 1.62000598
MAP3K1 28.977 28.884 7.845 6.428 -1.417 2.67029273
MAPK1 24.682 25.320 3.550 2.864 -0.686 1.60881553
MAPK10 29.584 30.501 8.452 8.045 -0.407 1.32592538
MAPK3 24.752 25.317 3.620 2.861 -0.759 1.69231810
MAPK8 26.054 26.241 4.922 3.785 -1.137 2.19923388
MAPK9 24.017 24.856 2.885 2.400 -0.485 1.39958460
MRAS 28.351 28.054 7.219 5.598 -1.621 3.07587821
MUC1 32.921 33.917 11.789 11.461 -0.328 1.25527288
MYC 21.336 20.368 0.204 -2.088 -2.292 4.89734494
NCK1 27.865 28.062 6.733 5.606 -1.127 2.18403942
NFKB1 25.900 26.377 4.768 3.921 -0.847 1.79875439
NKFB2 25.833 26.565 4.701 4.109 -0.592 1.50733392
NRAS 23.531 24.474 2.399 2.018 -0.381 1.30224293
PAK1 26.866 27.194 5.734 4.738 -0.996 1.99446026
PDPK1 25.271 24.939 4.139 2.483 -1.656 3.15141584
PIK3C2A 26.874 27.975 5.742 5.519 -0.223 1.16715775
PIK3C2B 28.366 27.840 7.234 5.384 -1.850 3.60499803
PIK3CA 30.151 31.074 9.019 8.618 -0.401 1.32042198
PIK3CB 27.331 27.618 6.199 5.162 -1.037 2.05195451
PIK3CD 29.771 30.281 8.639 7.825 -0.814 1.75807811
PIK3R1 25.395 24.925 4.263 2.469 -1.794 3.46775175
PIK3R2 23.930 24.400 2.798 1.944 -0.854 1.80750556
PLCG1 26.669 26.874 5.537 4.418 -1.119 2.17196293
PRKCA 27.060 27.737 5.928 5.281 -0.647 1.56590789
PRKCB 28.942 29.084 7.810 6.628 -1.182 2.26890972
PRKCD 24.436 24.707 3.304 2.251 -1.053 2.07483914
PRKCE 29.286 29.232 8.154 6.776 -1.378 2.59907515
PRKCG 32.547 33.169 11.415 10.713 -0.702 1.62676016
PRKCQ 29.408 28.382 8.276 5.926 -2.350 5.09824385
PRKCZ 25.659 26.224 4.527 3.768 -0.759 1.69231586
PTK2 25.681 25.633 4.549 3.177 -1.372 2.58829084
PXN 25.190 25.527 4.058 3.071 -0.987 1.98205845
RAB5A 22.711 22.985 1.579 0.529 -1.050 2.07052875
RAC1 26.734 26.925 5.602 4.469 -1.133 2.19314229
RAF1 26.121 26.417 4.989 3.961 -1.028 2.03919519
RASA1 26.777 27.111 5.645 4.655 -0.990 1.98618467
REL 26.517 26.868 5.385 4.412 -0.973 1.96291754
RELA 24.532 24.436 3.400 1.980 -1.420 2.67585171
RELB 31.068 32.400 9.936 9.944 0.008 0.99446925
39
RHOA 20.151 20.779 -0.981 -1.677 -0.696 1.62000598
RHOB 27.383 27.595 6.251 5.139 -1.112 2.16145007
RHOC 25.329 26.237 4.197 3.781 -0.416 1.33422350
RHOG 25.919 27.114 4.787 4.658 -0.129 1.09353523
RND3 25.525 24.759 4.393 2.303 -2.090 4.25747555
RRAS 30.739 31.758 9.607 9.302 -0.305 1.23541889
RRAS2 25.837 25.684 4.705 3.228 -1.477 2.78369139
SHC1 26.813 27.579 5.681 5.123 -0.558 1.47222549
SHC3 33.917 33.811 12.785 11.355 -1.430 2.69446416
SOS1 25.006 25.276 3.874 2.820 -1.054 2.07627977
SOS2 26.896 27.454 5.764 4.998 -0.766 1.70054695
SRC 26.203 26.169 5.071 3.713 -1.358 2.56329210
STAT1 26.415 26.758 5.283 4.302 -0.981 1.97383432
STAT3 26.338 27.060 5.206 4.604 -0.602 1.51781849
VAV1 28.362 28.919 7.230 6.463 -0.767 1.70172544
VAV2 26.457 26.227 5.325 3.771 -1.554 2.93630301
VAV3 27.880 28.312 6.748 5.856 -0.892 1.85574475

Table 14: EGFR TaqMan assay for EGFR-eGFP-V96 and EGFR-eGFP-A96 array plates

TaqMan array 96-well FAST plate consists of 18S, GAPDH, HPRT1, GUSB reference
genes. HPRT1 expression level in the HEK293 is 51.9, 18S (or) RPS18 expression is 98.9, and the
GAPDH expression level in the HEK293 is 96.8, whereas GUSB expression in the HEK293 is 8.5.
Since 18S is highly expressed in HEK293 cells, I took 18S as a reference gene for calculations.
Based on prior characterizations, I used EGFR-eGFP-A96 used as a control and EGFR-eGFP-V96
used as a test sample. All the genes in the TaqMan array plate were upregulated in EGFR-eGFP-
V96 cell lines compared to EGFR-eGFP-A96 after thermal stimulation. I depicted all the genes in
the below table by the range of colors proportionate to gene expression to make it easy to visualize
this complex data and understand it briefly. The color and intensity of the boxes are used to
represent relative gene expression. Some highly expressed genes in EGFR-eGFP-V96 cell lines
include Fos, Jun, and Protein Kinase C (PRKCQ).
40
Genes in TaqMan array plate:

ABI1 AKT1 AKT2 AKT3 ARAF BRAF CAV1 CAV2 CBL
CDH1 CHUK CSK CTNNB1 DIRAS3 EGF EGFR ELK1 EPS8
ERBB2 FOS GAB1 GRB2 HRAS IKBKB IKBKE IKBKG JAK1
JAK2 JUN KRAS MAP2K1 MAP2K2 MAP2K4 MAP2K7 MAP3K1 MAPK1
MAPK10 MAPK3 MAPK8 MAPK9 MRAS MUC1 MYC NCK1 NFKB1
NKFB2 NRAS PAK1 PDPK1 PIK3C2A PIK3C2B PIK3CA PIK3CB PIK3CD
PIK3R1 PIK3R2 PLCG1 PRKCA PRKCB PRKCD PRKCE PRKCG PRKCQ
PRKCZ PTK2 PXN RAB5A RAC1 RAF1 RASA1 REL RELA
RELB RHOA RHOB RHOC RHOG RND3 RRAS RRAS2 SHC1
SHC3 SOS1 SOS2 SRC STAT1 STAT3 VAV1 VAV2 VAV3

4.51272 2.23922 1.36037 2.02791 1.38895 2.10964 1.84292 1.83655 1.68529
1.98756 1.37744 1.55186 2.15546 1.88818 2.76446 3.41290 2.17045 1.97109
1.79129 1561.805 2.87387 1.91719 1.71594 2.02510 2.59368 1.69936 2.12137
2.20228 18.70041 2.12431 1.59327 1.56808 1.59880 1.62000 2.67029 1.60881
1.32592 1.692318 2.19923 1.39958 3.07587 1.25527 4.89734 2.18403 1.79875
1.50733 1.302242 1.99446 3.15141 1.16715 3.60499 1.32042 2.05195 1.75807
3.46775 1.807505 2.17196 1.56590 2.26890 2.07483 2.59907 1.62676 5.09824
1.69231 2.588290 1.98205 2.07052 2.19314 2.03919 1.98618 1.96291 2.675851
0.99446 1.620005 2.16145 1.33422 1.09353 4.257475 1.23541 2.78369 1.472225
2.69446 2.076279 1.70054 2.56329 1.973834 1.517818 1.701725 2.93630 1.855744

Table 15: Fold change in gene expression of EGFR-eGFP-V96 cells compared to EGFR-
eGFP-V96 after thermal stimulation

color key (Score):
1 2 3 4 5 18 1500

41

Figure 23: Expression levels of various upregulated genes such as RELB,
RHOG, PIK3C2A, RRAS, MUC1, NRAS, PIK3CA, MAPK10

Figure 24: Expression levels of various upregulated genes such as RHOC, AKT2,
CHUK, ARAF, MAPK9, SHC1, NKFB2, STAT3

1.00
1.10
1.17
1.24
1.26
1.31
1.33 1.33
RELB RHOG PIK3C2A RRAS MUC1 NRAS PIK3CA MAPK10
Fold change
Genes
Relative gene expression
1.34
1.37
1.39
1.40
1.41
1.48
1.52
1.53
RHOC AKT2 CHUK ARAF MAPK9 SHC1 NKFB2 STAT3
Fold change
Genes
Relative gene expression
42

Figure 25: Expression levels of various upregulated genes CSK, PRKCA,
MAP2K2, MAP2K1, MAP2K4, MAPK1, MAP2K7, RHOA

Figure 26: Expression levels of various upregulated genes such as PRKCG, CBL,
PRKCZ, MAPK3, IKBKG, SOS2, VAV1, HRAS

1.56
1.57
1.58
1.60
1.61
1.62
1.63 1.63
CSK PRKCA MAP2K2 MAP2K1 MAP2K4 MAPK1 MAP2K7 RHOA
Fold change
Genes
Relative gene expression
1.64
1.69
1.70 1.70
1.71 1.71 1.71
1.73
PRKCG CBL PRKCZ MAPK3 IKBKG SOS2 VAV1 HRAS
Fold change
Genes
Relative gene expression
43

Figure 27: Expression levels of various upregulated genes such as PIK3CD,
ERBB2, NFKB1, PIK3R2, CAV2, CAV1, VAV3, DIRAS3

Figure 28: Expression levels of various upregulated genes such as GRB2,
REL, EPS8, STAT1, PXN

1.77
1.80
1.81
1.82
1.85
1.85
1.87
1.90
PIK3CD ERBB2 NFKB1 PIK3R2 CAV2 CAV1 VAV3 DIRAS3
Fold change
Genes
Relative gene expression
1.93
1.97
1.98
1.98
1.99
GRB2 REL EPS8 STAT1 PXN
Fold change
Genes
Relative gene expression
44

Figure 29: Expression levels of various upregulated genes such as RASA1,
CDH1, PAK1, IKBKB, AKT3, RAF1, PIK3CB

Figure 30: Expression levels of various upregulated genes such as RAB5A,
PRKCD, SOS1, BRAF, JAK1, KRAS, CTNNB1

2.00
2.00
2.01
2.04
2.04
2.05
2.06
RASA1 CDH1 PAK1 IKBKB AKT3 RAF1 PIK3CB
Fold change
Genes
Relative gene expression
2.08
2.09 2.09
2.12
2.13
2.14
2.17
RAB5A PRKCD SOS1 BRAF JAK1 KRAS CTNNB1
Fold change
Genes
Relative gene expression
45

Figure 31: Expression levels of various upregulated genes such as RHOB,
ELK1, PLCG1, NCK1, RAC1, MAPK8, JAK2

Figure 32: Expression levels of various upregulated genes such as AKT1,
PRKCB, SRC, PTK2, IKBKE, PRKCE, MAP3K1
2.17
2.18
2.18
2.20
2.21
2.21
2.21
RHOB ELK1 PLCG1 NCK1 RAC1 MAPK8 JAK2
Fold change
Genes
Relative gene expression
2.25
2.28
2.58
2.60 2.61 2.61
2.69
AKT1 PRKCB SRC PTK2 IKBKE PRKCE MAP3K1
Fold change
Genes
Relative gene expression
46

Figure 33: Expression levels of various upregulated genes such as RELA, SHC3,
EGF, RRAS2, GAB1, VAV2

Figure 34: Expression levels of various upregulated genes such as MRAS, PDK1,
EGFR, PIK3R1, PIK3C2B

2.69
2.71
2.78
2.80
2.89
2.95
RELA SHC3 EGF RRAS2 GAB1 VAV2
Fold change
Genes
Relative gene expression
3.09
3.17
3.43
3.49
3.63
MRAS PDPK1 EGFR PIK3R1 PIK3C2B
Fold change
Genes
Relative gene expression
47

Figure 33: Expression levels of various upregulated genes such as RND3,
ABL1, MYC, PRKCQ, JUN

Bar graphs showing expression levels of upregulated genes by qPCR. Out of all the genes
in the TaqMan array plate, Fos, Jun, PRKCQ genes are abundantly upregulated in EGFR-eGFP-
V96 cells without ligand.

4.28
4.54
4.92
5.13
18.80
RND3 ABI1 MYC PRKCQ JUN
Fold change
Genes
Relative gene expression
48
CHAPTER FOUR: DISCUSSION

We hypothesize that thermally responsive ELP protein-polymer could replace the
endogenous ligands and activates various downstream signaling. Our previous lab researcher Zhe
Li developed a switch to activate with heat and target the EGFR signaling pathway. However, we
couldn’t evaluate many downstream signaling effects with transiently transfected cells because
only a small subset of the cells in an experiment are transfected. Since we wanted to study
downstream effects, we desired to produce highly transfected stable cell lines. If we measure
downstream signaling using qPCR, we may not be able to see a difference because only 10-20%
of cells are expressing, and we still have 80% of cells that cannot respond to temperature in a way
that EGFR-ELPs are. To solve this problem, we developed stable cell lines. Zhe Li already
developed temperature- sensitive variant. Stable temperature-insensitive cell lines were developed
by Hugo Avila using the FACS method.

EGFR-eGFP-A96 and EGFR-eGFP-V96 stable cell lines were characterized by western
blot for the presence of EGFR-eGFP-A96 and EGFR-eGFP-V96 proteins. The transitioning
temperature of EGFR-eGFP-A96 and EGFR-eGFP-V96 stable cell lines were characterized by
live-cell imaging. Before thermal stimulation, the EGFR fused V96 ELPs are soluble in an aqueous
solution, and EGFR signaling remains in “off” status. Temperature increase can activate the self-
assembly of EGFR fused V96 ELPs proteins that switch EGFR signaling to “on” status. This
accomplished EGFR activation without the presence of a ligand. Moreover, EGFR-ELPs act as
tunable switches, which is a convenient tool to quickly activate EGFR signaling and bring it back
down to study different EGFR pathways. In live-cell imaging, above the transition temperature
formation of clusters were observed at about 31ºC for temperature-sensitive variant. In contrast,
49
EGFR signaling remains in “off” status for temperature-insensitive variant due to lack of self-
association at any given temperatures.

The data showed that cells express EGFR-eGFP-ELP proteins and EGFR-eGFP-V96
transitions below physiological temperature while EGFR-eGFP-A96 doesn’t. Kinetic western blot
assay revealed temperature triggered activation of pERK1/2 without ligand for EGFR-eGFP-V96,
whereas EGFR-eGFP-A96 failed to show pERK1/2 activation after thermal stimulation. Kinetic
western blot data confirms temperature triggered activation of the RAS-RAF-MEK-ERK pathway.
Then we wanted to know various EGFR downstream signaling activation. As described earlier,
EGFR is known to activate various genes such as Myc, STAT, ABL1. I wanted to see fold change
for various genes activated by EGFR-V96. As expected, activation of various genes was observed
when EGFR-A96 heated. Based on the above data, we accomplished EGFR activation upon
heating. This indicates that we could replace the ligand to activate or inactivate EGFR rapidly.

50
CHAPTER FIVE: CONCLUSIONS

• Stably expressing cell lines were generated to get consistent and reliable downstream
signaling effects.
• EGFR-eGFP-V96 and EGFR-eGFP-A96 stable cell lines were characterized for the
presence of EGFR-eGFP-V96 and EGFR-eGFP-A96 proteins.
• EGFR-eGFP-V96 and EGFR-eGFP-A96 stable cell lines were characterized for Transition
temperature, and Tt of EGFR-V96 appears to activate EGFR signaling of ERK1/2.
• Kinetic western blot data revealed transient activation of EGFR. It shows that ELPs can be
used as intracellular switches to activate thermal stimulation rapidly.
• Various downstream expression levels known to depend on EGFR were used to confirm
EGFR activation differences between a temperature-sensitive and insensitive control. It
was evident that EGFR fused V96 ELPs upregulate many of the EGFR genes such as Fos,
Jun, Myc, and ABL1.

51
REFERENCES

1. Bethune G, Bethune D, Ridgway N, Xu Z. Epidermal growth factor receptor (EGFR) in
lung cancer: an overview and update. J Thorac Dis. 2010;2(1):48-51.
2. Yarden Y and Pines G (2012) The ERBB network: at last, cancer therapy meets systems
biology. Nat Rev Cancer 12, 553–563.
3. Burgess AW, Cho H‐S, Elgenblot C, Ferguson KM, Garrett TPJ, Leahy DJ, Lemmon
MA, Siwkowski MX, Ward CW & Yokoyama S (2003) An open‐and ‐shut case? Recent
insights into the activation of EGF/ErbB receptors Mol Cell 12, 541–552.
4. Ferguson KM. Structure-based view of epidermal growth factor receptor regulation. Annu
Rev Biophys. 2008; 37:353-373. Doi: 10.1146/annurev.biophys.37.032807.125829
5. Stamos J., Sliwkowski M.X., Eigenbrot C. Structure of the epidermal growth factor
receptor kinase domain alone and in complex with a 4-anilinoquinazoline inhibitor. J. Biol.
Chem. 2002; 277:46265–46272. Doi: 10.1074/jbc.M207135200.
6. Mitsudomi T, Yatabe Y. Epidermal growth factor receptor in relation to tumor
development: EGFR gene and cancer. FEBS J. 2010 Jan;277(2):301-8. Doi:
10.1111/j.1742-4658.2009.07448.x. Epub 2009 Nov 18. PMID: 19922469.
7. Li S, Schmitz KR, Jeffrey PD, Wiltzius JJ, Kussie P, Ferguson KM. Structural basis for
inhibition of the epidermal growth factor receptor by cetuximab. Cancer Cell. 2005
Apr;7(4):301-11. Doi: 10.1016/j.ccr.2005.03.003. PMID: 15837620.
8. Harris RC, Chung E, Coffey RJ. EGF receptor ligands. Exp Cell Res. 2003; 284:2–13.
9. Fukuoka, H.; Cooper, O.; Mizutani, J.; Tong, Y.; Ren, S.G.; Bannykh, S.; Melmed, S.
HER2/ErbB2 Receptor Signaling in Rat and Human Prolactinoma Cells: Strategy for
Targeted Prolactinoma Therapy. Mol. Endocrinol. 2011, 25, 92–103.
52
10. Wee, P.; Wang, Z. Epidermal Growth Factor Receptor Cell Proliferation Signaling
Pathways. Cancers 2017, 9, 52.
11. Pearson, G.; Robinson, F.; Beers Gibson, T.; Xu, B.E.; Karandikar,M.; Berman, K.;
Cobb,M.H.Mitogen-activated protein (MAP) kinase pathways: Regulation and
physiological functions. Endocr. Rev. 2001, 22, 153–183.
12. McCormick, F. (1998). Going for the GAP. Curr. Biol. 8, R673–R674.
13. Stokoe D, Macdonald, SG, Cadwallader K, Symons, M, Hancock JF. Activation of Raf as
a result of recruitment to the plasma membrane. Science 1994; 264:1463–7.
14. ZHANG, W., LIU, H. MAPK signal pathways in the regulation of cell proliferation in
mammalian cells. Cell Res 12, 9–18 (2002). https://doi.org/10.1038/sj.cr.7290105
15. Inoki K, Li Y, Xu T, Guan KL Rheb GTPase is a direct target of TSC2 GAP activity and
regulates mTOR signaling. Genes Dev. 2003; 17: 1829-1834
16. Proud CG. Control of the translational machinery by amino acids. Am J Clin Nutr 99(1):
231S-236S,2014.
17. O’Shea JJ, Holland SM, Staudt LM. JAKs and STATs in immunity, immunodeficiency,
and cancer. N Engl J Med. 2013; 368:161–70.
18. Hacein-Bey-Abina S, Le Deist F, Carlier F, et al. Sustained correction of X-linked severe
combined immunodeficiency by ex vivo gene therapy. N Engl J Med. 2002; 346:1185–93.
19. Berridge MJ, Irvine RF . (1989): Inositol phosphatase and cell signaling. Nature 341: 197–
205
20. Nishizuka Y . (1992): Intracellular signaling by hydrolysis of phospholipids and activation
of protein kinase C. Science 258: 607–613
53
21. Baldock C, Oberhauser AF, Ma L, Lammie D, Siegler V, Mithieux SM, Tu Y, Chow JYH,
Suleman F, Malfois M, Rogers S, Guo L, Irving TC, Wess TJ, Weiss AS. Shape of
tropoelastin, the highly extensible protein that controls human tissue
elasticity. Proceedings of the National Academy of Sciences. 2011; 108:4322–4327.
22. Partridge SM, Davis HF, Adair GS. The chemistry of connective tissues. 2. Soluble
proteins derived from partial hydrolysis of elastin. The Biochemical journal. 1955; 61:11–
21.
23. Meyer DE, Chilkoti A. Purification of recombinant proteins by fusion with thermally
responsive polypeptides. Nat Biotech. 1999; 17:1112–1115.
24. Despanie J, Dhandhukia JP, Hamm-Alvarez SF, MacKay JA. Elastin-like polypeptides:
Therapeutic applications for an emerging class of nanomedicines. J Control Release.
2016; 240:93-108. Doi: 10.1016/j.jconrel.2015.11.010
25. Wu, Y., Mackay, J. A., Mc Daniel, J. R., Chilkoti, A. & Clark, R. L. Fabrication of elastin-
like polypeptide nanoparticles for drug delivery by electro
spraying. Biomacromolecules. 10, 19–24 (2009).
26. Dan EM, Chilkoti A. Purification of recombinant proteins by fusion with thermally-
responsive polypeptides. Nat Biotechnol. 1999;17:1112.
27. Shi C, Meng Q, Wood DW. A dual ELP-tagged split intein system for non-
chromatographic recombinant protein purification. Appl Microbiol Biotechnol.
2013;97:829.
28. S.R. Macewan, W. Hassouneh, A. Chilkoti. Non-chromatographic purification of
recombinant elastin-like polypeptides and their fusions with peptides and proteins from
Escherichia coli JoVE, 88 (2014), p. 51583
54
29. K. Trabbic-Carlson, L. Liu, B. Kim, A. Chilkoti. Expression and purification of
recombinant proteins from Escherichia coli: comparison of an elastin-like polypeptide
fusion with an oligohistidine fusion Protein Sci., 13 (2004), pp. 3274-3284
30. J.A. Mackay, A. Chilkoti. Temperature sensitive peptides: engineering hyperthermia-
directed therapeutics Int. J. Hyperther., 24 (2008), pp. 483-495
31. Li Z, Tyrpak DR, Park M, Okamoto CT, MacKay JA. A new temperature-dependent
strategy to modulate the epidermal growth factor receptor. Biomaterials. 2018;183:319-
330. Doi:10.1016/j.biomaterials.2018.07.063
32. W. Liu, M.R. Dreher, D.Y. Furgeson, K.V. Peixoto, H. Yuan, M.R. Zalutsky, A. Chilkoti
Tumor accumulation, degradation, and pharmacokinetics of elastin-like polypeptides in
nude mice J. Contr. Release, 116 (2006), pp. 170-178

Abstract (if available)

Abstract Ligand-mediated EGFR forms homo or hetero dimers followed by autophosphorylation. After autophosphorylation, internalization occurs, which results in the activation of EGFR cell signaling. Previous researchers in the MacKay lab have designed ELP fusion proteins to modulate Epidermal Growth Factor Receptor signaling. Dr. Mackay抯 lab utilized the reversible phase behavior of ELP fusions to regulate cell signals. For example, the substitution of the ligand EGF by EGFR fused ELP can rapidly and transiently activate downstream signaling. Below its transition temperature, EGFR-V96 remains soluble. In contrast, above its transition temperature, EGFR-V96 fusions cluster, phosphorylate and internalize, initiate downstream kinase signaling. As a negative temperature-independent control, EGFR-A96 was demonstrated to avoid clustering at physiological temperature. Therefore, it has been selected as a control that does not activate signaling proteins to modulate gene expression. Ligand EGF, not only activates the EGF receptor, but it also activates other kinase receptors like HER2. Therefore, it was necessary to develop a tool to understand EGF receptors alone explicitly. Towards this hypothesis, Zhe developed a switch to activate EGFR alone in mammalian cells. This project seeks to understand whether EGFR fused ELPs are temperature sensitive. What biological effects do they have? Can EGFR-ELP fusions activates gene expression specific to EGFR signaling pathways? To answer these questions, downstream targets of EGFR were measured by western blot and RT-PCR to quantify gene expression of various EGFR signaling targets. This thesis concludes that EGFR-V96 specifically activates ERK1/2; furthermore, this is associated with a dramatic enhancement in nuclear transcription factors cFOS and JUN.

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Asset Metadata

Creator Boddu, Geetha (author)

Core Title Stable cell lines expressing elastin-like polypeptide fusions with epidermal growth factor receptor modulate gene expression in a heat dependent manner

Contributor Electronically uploaded by the author (provenance)

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Degree Conferral Date 2021-08

Publication Date 07/24/2021

Defense Date 07/21/2021

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

Tag EGFR,elastin-like polypeptide,ELP,epidermal growth factor receptor,OAI-PMH Harvest,temperature-sensitive

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Advisor John, Andrew MacKay (committee chair), Okamoto, Curtis (committee member), Seidler, Paul (committee member)

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