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Characterization of actin based motility in mammalian cells through LIM and SH3 domain protein 1 (LASP1) and elastin like polypeptide (ELP) fusion protein
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Characterization of actin based motility in mammalian cells through LIM and SH3 domain protein 1 (LASP1) and elastin like polypeptide (ELP) fusion protein
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Content
Characterization of Actin based motility in mammalian cells through LIM and
SH3 domain protein 1 (LASP1) and Elastin Like Polypeptide (ELP) fusion protein
by
Vishvesha Vaidya
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)
May 2020
Copyright 2020 Vishvesha Vaidya
ii
ACKNOWLEDGEMENTS
I am extremely grateful to my mentor Dr. Curtis Okamoto for giving me an opportunity to work
in his lab and gain research experience. He believed in my ability to take on an individual
project and allowed me the freedom to follow my interests. My path towards earning this
degree would have been far less rewarding without his encouragement and guidance. He
provided me with all the tools that I needed for my project and I will never forget the lessons
that I learned throughout my work in this lab.
I am very thankful to Anh Tan Truong and Taojian Tu who taught me all the necessary lab skills
and guided me with patience all the time. They taught me to think scientifically and logically
while performing every step of my project. I would not have completed my thesis without their
guidance. I would also like to thank Yutong Wang for guiding me in performance of cell culture
and western blotting and Yue Zhang for helping me order necessary enzymes and reagents.
Additionally, I extend my gratitude towards my committee members Dr. Andrew Mackay and
Dr. Ian Haworth for being a part of my thesis committee and for providing feedback on my
project. I also thank members of Mackay and Stiles lab for their help and support.
Finally, I would like to thank my parents for always believing in me. Without them, I never
would have been able to follow my passion of pursuing a Masters’ degree in pharmaceutical
sciences and complete my degree in good standing.
iii
TABLE OF CONTENTS
Acknowledgements.........................................................................................................................ii
LIST OF TABLES………………………………………………………………………………………………………………………….iv
LIST OF FIGURES....................……………………………………………………………………………………………………..v
Abstract…………………………………………………………………………………………………………………………………….vi
CHAPTER ONE: INTRODUCTION…………………………………………………………………………………………….....1
1.1 Mechanism of actin-based motility………………………………………………………………………….1
1.2 LASP1: Role in actin-based motility and association with cancer……………………………..3
1.3 ELP: Characteristics, applications and fusion proteins……………………………………………..6
CHAPTER TWO: MATERIALS AND METHODS…………………………………………………………………………….9
2.1 Plasmids to create a fusion protein between LASP1 with ELP V96…………………………..9
2.2 Restriction digest and ligation……………………………………………………………………………….16
2.3 Mammalian cell culture…………………………………………………………………………………………21
2.4 Transfection………………………………………………………………………………………………………….21
2.5 Bicinchoninic acid (BCA) assay………………………………………………………………………………22
2.6 Western Blotting…………………………………………………………………………………………………..22
CHAPTER THREE: RESULTS……………………………………………………………………………………………………….25
3.1 Linking of LASP1 to an oligonucleotide sequence …………………………………………………25
3.2 Fusion of LASP1 oligonucleotide plasmid DNA with ELP V96…………………………………31
CHAPTER FOUR: DISCUSSION………………………………………………………………………………………………….35
CHAPTER FIVE: CONCLUSION…………………………………………………………………………………………………..39
REFERENCES…………………………………………………………………………………………………………………………….41
iv
LIST OF TABLES
Table 1 Plasmid maps constructed for the experiment 9
Table 2 Reaction for Restriction digest of LASP1 16
Table 3 Reaction for Dephosphorylation of LASP1 Restriction digest 16
Table 4 Reaction for Restriction digest of oligonucleotide 17
Table 5 Ligation reaction of LASP1 with oligonucleotide 18
Table 6 Restriction digest of LASP1 oligonucleotide plasmid DNA 19
Table 7 Reaction for Restriction digest of ELP V96 19
Table 8 Reaction for blunting of ELP V96 Restriction digest with Klenow
fragment
20
Table 9 Reaction for ligation of LASP1-oligonucleotide plasmid DNA with
ELP V96
20
Table 10 Formula for preparation of lower gel 23
Table 11 Formula for preparation of stacking gel 23
Table 12 NANODROP readings for ligation of LASP1 with oligonucleotide 29
Table 13 NANODROP readings for restriction digest of LASP1-oligonucleotide
plasmid DNA
32
v
LIST OF FIGURES
Figure 1 Structure of lamellipodia, filopodia, stress fibers, cortex and focal
adhesions
2
Figure 2 Domain structure of LASP1 3
Figure 3 pCMV3-LASP1-C-Myc (6779 bp) 10
Figure 4 pCMV3-LASP1-C-Myc-oligonulceotide (6797 bp) 11
Figure 5 V96 ELP (6925 bp) 12
Figure 6 V96- LASP1 (8241 bp) 13
Figure 7 Oligonucleotide sequence 14
Figure 8 DNA gel for Restriction digest of LASP1 25
Figure 9 Transformation plates for ligation of LASP1 with oligonucleotide 27
Figure 10 DNA gel for diagnostic digests 30
Figure 11 DNA gel for Restriction digest of LASP1 oligonucleotide fusion
protein
31
Figure 12 DNA gel for Restriction digest of ELP V96 32
Figure 13 Colony seen on 5:1 ratio plate of LASP1-oligonucleotide plasmid
DNA and ELP V96 ligation reaction
33
vi
ABSTRACT
Actin-based motility is responsible for a variety of cell processes through which living cells may
change their morphology in response to environmental signals. This includes protrusions like
lamellipodia and filopodia or wrapping and ingesting of a particle through phagocytosis. The
assembly and disassembly of actin filaments and their organization into functional higher order
networks are regulated by a plethora of actin-binding proteins. LASP1 is one such versatile
actin-binding protein which plays an important role in actin motility and cell migration.
Additionally, it is currently being studied as a biomarker protein owing to its overexpression in a
variety of cancers. A fusion of LASP1 protein with an ELP was created through use of
recombinant DNA technology in an attempt to help define the role of LASP1 in actin-dependent
motility of intracellular organelles. ELPs are biopolymers which work on the principle of
temperature dependent phase transition and are being explored in the fields of cancer therapy,
protein purification and tissue scaffolding. The constructed LASP1- ELP fusion protein was
expressed in a mammalian cell line (HEK 293T) via transient transfection and was analyzed by
western blotting. Although, no further steps were performed in this particular project, it has a
potential to compare the effect of LASP1- ELP fusion protein with that of LASP1 alone on actin-
based intracellular motility and cancer metastasis and to determine if LASP1 can be silenced
with the help of ELP to reduce this metastasis. Alternatively, it can be determined whether
LASP-1 dependent modulation of actin-based motility can be activated by ELP self-assembly.
The model is that temperature-dependent ELP coacervates would form actin-dependent, motile
artificial organelles and provide a tool to characterize the mechanism of actin-based motility of
organelles.
1
CHAPTER ONE: INTRODUCTION
1.1 Mechanism of actin-based cell motility
Animal cells have the ability to change their shape for adapting to their environment, moving
through narrow spaces, dividing, or allowing exocytosis and endocytosis (Blanchoin et al.,2014).
These shape changes and migration are crucial for the functionality of body systems (Svitkina,
2018). The machinery for these shape changes relies on the assembly of proteins, in particular
actin which is a globular protein having ATPase activity (Blanchoin et al.,2014; Suetsugu et
al.,2004). Actin exists either as individual actin molecules referred to as globular actin (G-actin)
or it can self-assemble into filamentous structures with specific polarity called filamentous actin
(F-actin) through the process called polymerization, in an ATP-dependent fashion (Suetsugu et
al.,2004). The collection of actin filaments together with their regulatory, actin binding and
other accessory proteins forms the actin cytoskeleton. Forces generated by the actin
cytoskeleton power diverse motility processes of the cells. Regulatory proteins control all
aspects of actin filament dynamics in time and space including actin filament nucleation,
elongation, and disassembly while actin-binding proteins assemble most actin filaments into
networks and bundles adapted for specific tasks (Svitkina, 2018). These include lamellipodia,
filopodia and stress fibers. Lamellipodia are branched and crosslinked networks which push the
cell membrane by polarizing it. They thus form major engines of cell movement. Filopodia are
thin fingerlike structures filled with tight (closely packed) and parallel bundles of filamentous
actin. Cells make use of these filopodia as ‘tentacles’ or ‘antennae’ to probe (explore) their
microenvironment (Pieta et al.,2008). The microenvironment is the local environment
surrounding the cell which influences cellular behavior. Stress fibers are contractile actin
2
bundles that span the cell body in a direction which is parallel to the direction of movement.
Their function is to connect the cell cytoskeleton to extracellular matrix via focal adhesion sites,
which in turn provides traction for cellular motility. The cell cortex contains a thin layer of actin
which resides just underneath the plasma membrane at the sides and back of the cell and is
important for cell shape maintenance and cell shape changes (Blanchoin et al.,2014). Actin
based cell motility can also play important roles in various diseases. It enhances invasion and
metastasis of cancer/tumor cells and leads to chronic inflammatory diseases through migration
of immune cells to tissues (Svitkina, 2018). Some microbial pathogens manipulate motility
mechanisms of host cells in order to avoid immune surveillance and also to favor their own cell-
to-cell spread (Svitkina, 2018).
Figure 1: Structures of specific components of F-actin organization (Blanchoin et al.,2014). The figure shows lamellipodia which
are branched and crosslinked filaments helping in cell movement. Filopodia are finger like structures to help the cell probe its
microenvironment. Stress fibers are contractile actin bundles. Focal adhesions help in attachment of of stress fibers to the
extracellular matrix. The cortex is a thin actin layer that helps in cell shape maintenance.
3
1.2 LASP1: Role in actin-based motility and association with cancer.
LASP1 is a relatively widely distributed but differentially expressed actin-binding protein which
belongs to the nebulin family of actin-binding proteins (Zhang et al.,2009). It is expressed
ubiquitously in normal tissues at low level, but its expression is enriched in certain actin-rich cell
and tissue types. The gene encoding LASP1 protein was cloned around two decades ago from a
cDNA library of breast cancer metastases (Orth et al.,2015). LASP1 is the first protein of the
class consisting of one N-terminal LIM (explanation is below) and one terminal Src Homology 3
(SH3) domain, thus founding a new LIM protein subfamily of the nebulin group (Orth et
al.,2015).
Figure 2: Domain structure of LASP1 (Butt et al.,2018). The LIM and SH3 domains are shown in red and green colors
respectively. The LIM domain is an adaptor domain for multimeric protein complexes whereas the SH3 domain binds to a
variety of motility-associated proteins. The nebulin-like repeats are shown in blue which display a conserved motif involved in
binding to F-actin. The magenta color displays the phosphorylation sites Serine 146 and Tyrosine 171. The proteins binding to
each respective domain are shown in black below LASP1.
4
The N-terminal LIM (lin-11, Isl-1, and mec-3) domain (residues 5–57) is rich in cysteine and is
composed of two zinc finger domains. It functions as an adaptor for multimeric protein
complexes. The zinc domains were originally observed in three proteins lin-11, Isl-1, and mec-3
and so the zinc motif derives its name from the first letters of these proteins (Lin/Isl/Mec). It
was revealed from a solution structure that the flexible hydrophobic core of the LIM1 domain
provides a binding interface which favors binding of its physiological targets. For example,
Figure 2 shows binding of the zinc finger LIM domain to CXC (two cysteine residues with an
interspersed amino acid) chemokine receptors 1-4. It is predicted that binding of CXCR1-3
requires phosphorylation at Serine 146 while binding of CXCR4 is independent of
phosphorylation of LASP1. Recently LASP1 has also shown to bind Ubiquitin Like with PHD And
Ring Finger Domains 1 (UHRF1) through the LIM domain and together they function as a hub for
epigenetic machinery (Butt et al.,2018). Nebulin like repeats (NR1 and NR2) display a
conserved motif and are known to interact directly with F-actin as well as with Kelch-related
Protein 1 (KRP1) involved in pseudopodial elongation (Butt et al.,2018; Orth et al.,2015). A
linker region following the nebulin repeats consists of two phosphorylation sites namely Serine
146 and Tyrosine 171. (Butt et al.,2018) As per recent studies, the PKA and PKG mediated
phosphorylation at Serine 146 reduces binding of LASP1 to F-actin, increases translocation of
the protein to the cytosol and also leads to decrease in migration of cells. At Tyrosine 171, the
phosphorylation by Abelson tyrosine kinase blocks LASP1 translocation to focal complexes. This
occurs in cells which are apoptotic. The phosphorylation of LASP1 by Src kinase plays a role
inside platelets by causing translocation of LASP1 to focal contacts, as well as cytoskeleton
rearrangement and activation of these cells (Mihlan et al., 2013). The SH3 domain i.e SRC
5
homology 3 domain is located at the C-terminal of LASP1 structure. Through this domain, lasp1
interacts with a large number of motility-associated proteins like zyxin, dynamin, lipoma
preferred partner (LPP), actin, palladin and Krp1 (Mihlan et al., 2013). This interaction of LASP1
with motility-related proteins is consistent with the involvement of LASP1 in reorganization of
the actin cytoskeleton during cellular motility. It is also involved in localization to cellular
protrusions like focal adhesions, lamellipodia, and filopodia, or pseudopodia leading to cell
migration (Ruggieri et al.,2017). Thus, we can say the unique domain composition renders
LASP1 to be an extremely versatile, modular scaffolding protein and is indicative of its
implications in various cellular functions including cellular motility, cell migration and cell
signaling (Orth et al.,2015; Ruggieri et al.,2017). However, alongside playing numerous roles
under normal physiological conditions, LASP1 protein also plays a role in human cancers by
affecting tumor aggressiveness (Orth et al.,2015). It appears to regulate cancer cell metastatic
propensity by disturbing the architecture and dynamics of F-actin, triggering cell migration and
invasion (Ruggieri et al.,2017). Data show that it can undergo nuclear localization or
translocation in cancer cells in the same way as it is localized in normal cell’s cytosol by
interacting with actin cytoskeleton and other actin-binding proteins like zyxin, Vasodilator-
stimulated phosphoprotein (VASP), LPP, and many more (Ruggieri et al.,2017). The siRNA
mediated LASP1 knockdown showed decreases in migration of Bt-20 and MCF-7 breast cancer
cell lines, as well as decreases in cell proliferation. Similar results concerning inhibition of cell
migration and invasion were obtained from ECA109 and KYSE510 cell lines thus providing
evidence for LASP1 mediated aggressiveness in cancer (Orth et al.,2015). Another study showed
colocalization of LASP1 with actin in cell membrane extensions of human BT-274 breast and SK-
6
OV-3 ovarian cell lines (Schreiber et al., 1998). Thus, LASP1 has clinical significance and
potential value as a new cancer diagnostic biomarker (Ruggieri et al.,2017). There is a study
that LASP1 may be a biomarker for urinary bladder cancer (Sato et al., 2017).
1.3 ELPs: Characteristics, applications and fusion proteins
Natural elastin is one of the most abundant fibrous proteins in the extracellular matrix of
vertebrates. It is the prevailing constituent of mature elastic fibers and is involved in providing
elasticity to flexible tissues. It also acts as a signaling protein modulating cell matrix interactions
alongside performing the mechanical and structural role (Rodríguez-Cabello et al.,2016).
Elastomeric polypeptides are protein-based polymers which are becoming popular in various
biomedical applications due to the increasing demand for molecules having strictly specified
properties. ELPs are artificial, genetically encodable biopolymers belonging to the class of these
elastomeric proteins (Kowalczyk et al., 2014). As a result of being genetically encodable, they
can be manipulated by using molecular cloning techniques and can be synthesized in
heterologous hosts like E. Coli. The structure of an ELP is a pentameric sequence of (Valine-
proline-glycine-Xaa- glycine) n where Xaa can be any amino acid and ‘n’ specifies the number of
times the sequence is being repeated (Peddi et al., 2018). ELPs undergo temperature-
dependent phase separation which is reversible and a quite rapid process. Below the phase
transition temperature (Tt), the ELPs are highly soluble but when the temperature is above Tt,
ELPS assemble into an aqueous two-phase system that is they phase separate into a gel-like
coacervate (Pastuszka et al., 2012; Peddi et al., 2018). Tt can be genetically controlled by
bringing a modification in properties of Xaa and n (Pastuszka et al.,2012). If Xaa is a
7
hydrophobic guest residue like valine or isoleucine, the Tt is lower, but if it is a hydrophilic guest
residue the Tt is higher. For ELPs with larger molecular weights that is higher number of ‘n’
repeats, the Tt is lower and vice versa. Thus, these two parameters are very important in
modifying Tt such that the temperature dependent phase separation can occur at
physiologically relevant temperatures and also at reasonable concentrations after ELPs are
expressed in the cytoplasm (Pastuszka et al.,2012). The unique behavior of ELPs can be further
triggered by a number of environmentally based stimuli like temperature, pH or ionic strength
and is dependent considerably on concentration of ELP in solution form (Kowalczyk et al.,
2014). ELPs can be purified non-chromatographically from the recombinant expression systems
by using multiple rounds of inverse phase transition cycling because of their unique
temperature-dependent transition property (Peddi et al., 2012). This property of ELPs also
favors their applications in a variety of areas like drug delivery, tissue engineering, as vaccine
carriers and therapeutic proteins (Fletcher et al.,2019). The biodegradability and
biocompatibility with human blood, tissues and tissue fluids as well as lack of immunogenicity
make ELPs an ideal candidate for these applications. Moreover, ELPs retain their thermal
responsiveness even after genetic fusion with soluble proteins and peptides. While soluble, the
fusion polymers of ELPS have an intracellular diffusion coefficient which is similar to cytosolic
proteins and above Tt they form distinct microdomains with a reduced diffusion coefficient
(Pastuszka et al.,2012). But, in such cases the transition temperature of parent ELPs is affected
by the ‘fusion ΔTt effect’ which is the transition temperature difference between fusion protein
and parent ELP. As a result, it is necessary to make a rational choice of an ELP sequence and
chain length for achieving the desired Tt for a specific protein ELP fusion (Hassouneh et al.,
8
2012). ELP-anticancer drug fusion proteins are nowadays explored in cancer treatment where
they are injected in conjunction with external mild hyperthermia to the tumor. This promotes
coacervation of ELPs and increase in concentration in the tumor vasculature stimulating the
effect of fusion protein at the tumor site (Mcdaniel et al.,2010). The thesis project was aimed at
using this thermal responsive behavior of ELP fusion proteins to determine whether the LASP1-
ELP fusion protein can behave as endogenous LASP1 and if it can have an effect on actin-based
intracellular motility similar to LASP1 alone. In addition, another model that can be tested is
that the temperature-dependent coacervates of LASP1-ELP fusion proteins would form actin-
dependent, motile artificial organelles, as determined by an increase in diffusion coefficient of
the coacervates and provide a tool to characterize the mechanism of actin-based motility of
organelles.
9
CHAPTER TWO: MATERIALS AND METHODS
2.1 Plasmids to create a fusion protein between for LASP1 and ELP V96.
Plasmids are small circular pieces of DNA which replicate independently from the host’s
chromosomal DNA. These are found mainly in bacteria but also in some eukaryotes. They
consist of origin of replication, antibiotic resistance gene, multiple cloning site, promoter region
and a few others which favor their application in recombinant DNA technology. They are often
constructed in labs using various cloning techniques and transformed into bacteria (Monroe,
2014). For this project, plasmid maps were constructed using Snap gene for LASP1 wild type,
LASP1 with oligonucleotide, ELP V96 and fusion protein of LASP1-oligo with ELP V96. Unique
cutter enzymes are shown in the plasmid maps’ figures below. An additional linkage of LASP1
with an oligonucleotide sequence (Figure 7) was performed for the purpose of avoiding
frameshift mutation.
Table 1: Plasmids maps constructed for the experiment.
Components Plasmid map
LASP1 wild type Figure 3
LASP1 with oligonucleotide Figure 4
ELP V96 Figure 5
LASP1-oligo with ELP V96 Figure 6
10
Figure 3: pCMV3-LASP1-C-Myc (6779 bp)
11
Figure 4: pCMV3-LASP1-C-Myc-oligonulceotide (6797 bp)
12
Figure 5: V96 ELP (6925 bp)
13
Figure 6: V96- LASP1 (8241 bp)
14
Figure 7: Oligonucleotide sequence
15
The plasmid for LASP1 wild type was ordered from Sino Biologicals, oligonucleotide sequence
from Integrated DNA Technologies (IDT) and the ELP V96 sequence from Dr. Mackay’s
laboratory at USC School of Pharmacy. The longer repeats of ELP show lower transition
temperatures, so ELP V96 was chosen for the construct to make sure the fusion protein can
transition at a reasonable physiological temperature. The LASP1 plasmid obtained was
resuspended as per the protocol on the plasmid data sheet and the concentration obtained
after resuspension was 0.1 µg/µl. It was then transformed into E. coli cells using the protocol
for One Shot Top 10 E. Coli cells (https://assets.thermofisher.com/TFS-
Assets/LSG/manuals/oneshottop10_chemcomp_man.pdf). The cells were ordered from the
company Thermo Fisher Scientific located in Waltham, MA. The colonies obtained were used
for performing maxipreps as per the Qiagen protocol and concentration of DNA after Maxiprep
was found to be 2817.33 ng/µl that is 2.8µg/µl. This sample was then diluted to a new
concentration of 0.56 µg/µl for the ease of further reactions and calculations. The sample of
oligonucleotide was centrifuged at full speed in a microfuge for 10 seconds and a 100 µM stock
solution of oligonucleotide was prepared as per the protocol on the data sheet. 40 µl of double
distilled water was added to 10 µl of solution from the stock, and the resulting 50 µl was
incubated at 95 ℃ for 3 min. It was then allowed to cool to room temperature and stored at 4 ℃
for use in further experiments. The working concentration of oligonucleotide was 0.17 µg/µl.
The remaining oligonucleotide stock solution was stored at -20 ℃.
16
2.2 Restriction digest and ligation
Restriction digest (RD) is a process where restriction enzymes are used to cut open a plasmid
(backbone) and insert a linear fragment of DNA (insert) which is also cut by compatible
restriction enzymes (Ford, 2016). Nowadays high-fidelity enzymes are used which favor
digestion in a short period of time. Restriction digest of LASP1 was performed using NEB Cloner.
Table 2: Reaction for Restriction digest of LASP1.
Component Quantity (in µl)
DNA 3.55
10X Cut smart Buffer 5
Hind III HF 2
Nuclease-Free (NF) water 39.45
Total reaction volume 50
The reaction was then incubated at 37 ℃ for 1 hour. It was then heat inactivated at 80 ℃ for 20
min as per NEB Cloner guidelines (https://nebcloner.neb.com/#!/). Heat inactivation of
Restriction digest was followed by a dephosphorylation reaction. To the above reaction (table
2) the following were added:
Table 3: Reaction for dephosphorylation of LASP1 Restriction digest.
Component Quantity (in µl)
Antarctic Phosphatase buffer 5.5
Antarctic phosphatase enzyme 2
17
The reaction was then incubated at 37 ℃ for 1 hour. The oligonucleotide was first annealed that
is heated at 95 ℃ for 3 min and cooled to room temperature before use. The Restriction digest
for oligonucleotide was then performed as per following formula:
Table 4: Reaction for Restriction digest of oligonucleotide.
Component Quantity (in µl)
DNA 11.9
10X Cut smart Buffer 5
Hind III HF 2 l
NF water 31.1
Total reaction volume 50
The reaction was then incubated at 37 ℃ for 1 hour followed by heat inactivation at 80 ℃ for 20
min. Ligation of LASP1 Restriction digest mixture (vector) and oligonucleotide Restriction digest
mixture (insert) was performed as per quantities obtained from NEBioCalculator. Ligation
makes use of a DNA ligase enzyme to anneal the vector and insert covalently at the expense of
ATP (Ford, 2016). Concentration obtained from NANODROP was used for determining
quantities of vector and insert in microliters (µl) in correspondence to NEBioCalculator values.
Ligation reaction for different ratios was carried out as follows:
18
Table 5: Ligation reaction of LASP1 with oligonucleotide.
Component Insert:vector ratio (amounts in µl)
3:1 5:1 7:1 20:1 100:1
T4 DNA ligase buffer 2 2 2 2 2
Vector DNA (56 ng) 2 2 2 2 2
Insert DNA 1.03 1.7 2.4 6.9 34.5
NF water 14 13.3 12.6 8.1 7.5
T4 DNA ligase 1 1 1 1 1
Total reaction volume 20 20 20 20 20
A negative control was prepared for further experiment by repeating the reaction for 7:1 ratio
without the T4 DNA ligase. The mixtures were then incubated at 16 ℃ overnight in the PCR
machine. Ligation reaction was followed by transformation where positive control (intact LASP1
plasmid) and negative control (7:1 ratio without T4) were maintained. Then 7:1-1 and 20:1-2
ratios from table 12 were used for further reactions. Restriction digest was performed in
replicates of 2 for both the ratios.
19
Table 6: Restriction digest of LASP1-oligonucleotide plasmid DNA.
Component Quantity (in µl)
7:1-1 (2 tubes) 20:1-2 (2 tubes)
DNA (2 µg) 7.2 6.3
10X Cut smart Buffer 5 5
AfeI 2 2
NF water 35.8 36.7
The reaction was incubated at 37 ℃ for 2 hours followed by dephosphorylation. Restriction
digest for V96 (ELP) was performed as follows:
Table 7: Reaction for Restriction digest of ELP V96.
Component Quantity (in µl)
DNA 6.66
10X Cutsmart buffer 2.5
NdeI 1
ACuI 1
SAM 0.1
NF water 13.74
Total reaction volume 25
The reaction was incubated for 1 hour at 37 ℃ and heat inactivated at 65 ℃ for 20 min. Gel
electrophoresis was performed to get the desired band and DNA was then extracted from the
band using Qiagen protocol for gel extraction. The blunting reaction with Klenow fragment was
done as follows:
20
Table 8: Reaction for blunting of ELP V96 Restriction digest with Klenow fragment.
Component Quantity in µl
DNA (383.6 ng) 28
T4 DNA ligase buffer 5
dNTPs 0.2
Klenow 0.2
NF water 16.6
Total volume 50
The reaction was then incubated at room temperature for 15 minutes. 1µl of 0.5M EDTA
solution was added (to 10 mM) and the mixture was incubated again at 75 ℃ for 20 min. PCR
purification was performed instead of gel electrophoresis and the NANODROP reading obtained
was used in further ligation calculations. The ligation of LASP1-oligo with V96 ELP was
performed as follows:
Table 9: Reaction for ligation of LASP1-oligonucleotide plasmid DNA with ELP V96.
Component Quantities (in µl)
3:1 5:1 7:1 20:1
T4 DNA ligase buffer 2 2 2 2
Vector DNA (50 ng) 2.2 2.2 2.2 2.2
Insert DNA 1.2 2.01 2.82 8.06
NF water 13.7 12.8 12.03 6.8
T4 DNA ligase 1 1 1 1
Total reaction volume 20 20 20 20
21
The reaction tubes were then incubated at 16 ℃ in the PCR machine overnight. Transformation
was performed using 3 µl of DNA, TB broth in place of SOC medium and 150 µl was used for
spreading on the plates. The single colony obtained on a 5:1 ratio plate was used for further
miniprep procedure.
2.3 Mammalian cell culture.
Mammalian cell culture is one of the important pillars of life sciences where the cells originally
isolated from animal tissues are grown in vitro in a dish or flask (Greb, 2017). The cell line used
for this project was HEK293T. This cell line was obtained from Dr. Stiles lab at USC School of
Pharmacy. ATCC product sheet for HEK293T was used for carrying out the ‘Handling procedure
of frozen cells’ as well as for the sub culturing procedure. Cells were subcultured 3 times using
the protocol from the same product sheet.
2.4 Transfection
Transfection is the process where nucleic acids (DNA or RNA) are introduced into cells by
artificial means other than viral infection. These include various chemical, biological or physical
methods resulting in a change in a cell’s properties allowing the study of gene function and
protein expression in the context of the cell.
(https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-
basics/transfection-basics/introduction-to-transfection.html) Transient transfection was
performed using Lipofectamine 3000 Reagent Protocol. Different amounts of cells were plated
in each well of the 6-well plate during the seeding step that is 100,000; 200,000; 300,000;
22
400,000; 500,000 and 600,000 cells in plates 1 to 6 respectively. Transfection was performed on
the last three wells with 400,000; 500,000 and 600,000 cells since these three wells showed
desired level of confluency. The transfected cells were collected from the last three wells using
RIPA lysis buffer protocol. 200 µl of RIPA buffer and 6 µl of protease inhibitor was used for each
of the last 3 wells and centrifugation was carried out at 900 rpm for 3 min. At the end of the
procedure the samples were stored at -20 ℃ and a BCA assay was performed.
2.5 Bicinchoninic Acid (BCA) assay
BCA assay protocol was used to determine the concentration of protein in samples. The assay
was performed using the microplate method. The sample was diluted twice (25 µl of sample
plus 25 µl of distilled water) before using in the assay and absorbance was measured at 562nm
in a spectrophotometer. The concentration of protein in the sample was then determined by
using the standard curve by measuring the absorbance of a series of known concentrations of
Bovine Serum Albumin (BSA).
2.6 Western blotting
Western blotting is a technique used to identify a specific protein from a complex mixture of
proteins extracted from a cell. This is accomplished by using three elements namely separation
by size, transfer onto a solid support and marking the target protein with a primary as well as
secondary antibody to visualize (Liu et al.,2014). For this project, western blotting was
performed using 10% resolving gel and 4% stacking gel. Lower buffer needed for lower gel was
prepared using 1.5M Tris and 10% SDS, and pH was adjusted to 8.8. Stacking buffer needed for
23
stacking gel was prepared using 0.5M Tris with 0.4% SDS and pH was adjusted to 6.8. The gels
were prepared as follows:
Table 10: Formula for preparation of 10% resolving gel.
Ingredients Quantities
Water 4 ml
4x lower buffer 2.5 ml
Acrylamide monomer 3.4 ml
10% sodium dodecyl sulfate (SDS) 100 µl
10% Ammonium Persulfate (APS) 40 µl
Tetramethyl ethylenediamine (TEMED) 8 µl
Table 11: Formula for preparation of stacking gel.
Ingredients Quantities
Water 2.18 ml
4x stacking buffer 1.25 ml
Acrylamide monomer 533 µl
10% SDS 40 µl
10% APS 40 µl
TEMED 8 µl
Transfer was carried out for 90 min at 4 ℃ instead of overnight transfer. The anti-Myc Tag
antibody was used as a primary antibody because the plasmid map for LASP1-V96 fusion
protein contained a Myc tag for the fusion protein to be used to confirm expression of the
fusion protein in cells. The secondary antibody used was conjugated to horseradish peroxidase
24
(HRP) as it results in a strong signal in a short span of time leading to increase in the detection
of the target molecule. Imaging of the membrane was performed after incubating the
membrane in ECL (Enhanced chemiluminescence) mix for 1 min.
Flowchart for quick review of Materials and methods employed in this project:
Restriction digest of LASP1 wild type using Hind III HF followed by
dephosphorylation using Antarctic Phosphatase
Restriction digest of oligonucleotide using Hind III HF
Ligation of LASP1 wild type with oligonucleotide using T4 DNA ligase
Restriction digest of LASP1-oligonucleotide plasmid DNA using AfeI
Restriction digest of ELP V96 using NdeI and AcuI along with SAM cofactor
Blunting of ELP V96 Restriction digest mixture using Klenow fragment
Ligation of LASP1-oligonucleotide plasmid DNA with ELP V96
Transient transfection of ligated product of LASP1-oligonucleotide and ELP
V96 into HEK293T mammalian cell line
Collection of transfected cells using RIPA lysis buffer followed by BCA assay
to determine protein concentration of transfected cells
Analyzing the expression of fusion product of LASP1-oligonucleotide and ELP
V96 from the transfected cells using western blotting. Anti-Myc tag and anti-
rabbit HRP used as primary and secondary antibodies respectively.
25
CHAPTER THREE: RESULTS
3.1 Linking of LASP1 to an oligonucleotide sequence
The Restriction digest mixture of LASP1 with Hind III HF after dephosphorylation was analyzed
through agarose gel electrophoresis. It is a technique which is used for separation of DNA
fragments varying in size from 100 bp to 25 kb. The negatively charged DNA molecules move
towards the positive anode when placed in an electric field and get separated by size on the
agarose gel. In this project, 1% agarose was used for preparing the DNA gel (Lee et al.,2012).
SYBR safe was used for staining of the gel. Loading dye was added to the Restriction digest
mixture, and the sample was divided into two parts to be loaded into two wells of the gel. The
gel was run at 100V for 1 hour and analyzed under UV light. Two bands were obtained
corresponding to two wells on the gel.
Figure 8: DNA gel for Restriction digest of LASP1. The bands obtained in lanes 1 and 2 in the figure both describe the restriction
digest of LASP1 with Hind III HF. As the sample was divided into two parts while loading on the gel, there are two bands
visualized.
1 2
26
The two bands were cut out with a sharp blade and was purified using the Qiagen protocol for
gel extraction. NANODROP 2000 was used to determine the concentration of purified DNA.
Readings obtained were 26.2 ng/µl and 28.1 ng/µl for band 1 and band 2 respectively which
were needed to be identified on the gel. Restriction digest of oligonucleotide was subjected to
PCR purification instead of gel electrophoresis. This was done because the small size of
oligonucleotide (28 bp) cannot be detected on the DNA gel. The NANODROP reading of 33.5
ng/µl of DNA was obtained after PCR purification. The NANODROP readings obtained were used
for determining amounts of vector and insert in microliters for the ligation reaction (Table 4).
The success of ligation was determined by performing transformation and analyzing the
colonies. Transformation is a process in which foreign DNA is introduced inside the bacterial
cell. Plasmids are transformed into bacteria because bacteria act as a good means for storing
and replicating plasmids (https://www.addgene.org/protocols/bacterial-transformation/).
Competent E. coli cells are commonly used for obtaining high transformation efficiency.
27
Figure 9: Transformation plates for ligation of LASP1 with oligonucleotide. The figure shows colonies obtained on the
transformation plates for various ligation ratios of LASP1 and oligonucleotide. The plates have been numbered from 1 to 7. The
plates 1 to 5 show different insert: vector ligation ratios. Plate 1 is ratio 3:1; plate 2 is ratio 5:1; plate 3 is ratio 7:1; plate 4 is
ratio 20:1 and plate 5 is ratio 100:1. Plates 6 and 7 represent positive (intact LASP1 plasmid) and negative (Reaction for 7:1 ratio
without T4 DNA ligase) controls respectively.
1 2 3
4 5
6
7
28
Colonies were obtained for all the ratios and for the positive control. Few colonies were also
seen on the negative control plate. This is unusual but might have happened because of some
undigested vector may have transformed the host bacteria. Three colonies each from ratios 3:1,
5:1 and 7:1 while 2 colonies each from ratios 20:1 and 100:1 were picked. These were then
added to 5ml TB broth containing kanamycin antibiotic and incubated at 37 ℃ and 250 rpm in a
shaking incubator, room temperature, overnight (that is for 16 hours) The solution in all the
tubes became turbid indicating growth of bacteria. Minipreps were then performed using 3ml
of culture from each tube and 50 µl DNA elute collected for each. NANODROP 2000 was used to
determine the concentration of DNA obtained from minipreps. 2 µl of sample was used for each
determination. The readings from NANODROP were as follows:
29
Table 12: NANODROP readings for ligation of LASP1 with oligonucleotide.
Ligation Ratio NANODROP reading (in ng/µl)
3:1-1 317.1
3:1-2 211.76
3:1-3 281.9
5:1-1 274.86
5:1-2 244.93
5:1-3 250.16
7:1-1 286.56
7:1-2 277.63
7:1-3 288.53
20:1-1 322.43
20:1-2 317.06
100:1-1 318.5
100:1-2 329.26
30
Diagnostic digests were performed for the ligation mixtures using 0.5 µg of DNA; 10X Cut smart
buffer; AfeI, NotI-HF restriction enzymes and the final volume in each reaction was adjusted to
25 µl using NF water. The samples were then run on a DNA gel. These steps were done to check
if the ligation product obtained was the correct one or not through the band size obtained on
the gel.
Figure 10: DNA gel for diagnostic digests of LASP1 oligonucleotide ligation reaction. The lanes are numbered from 1 to 13. Each
of the ligation ratios used for linking LASP1 with oligonucleotide was subjected to a diagnostic digest using AfeI and NotI HF
enzymes. The insert: vector ligation ratios corresponding to each lane were as follows:
Lane 1=3:1-1; lane 2=3:1-2; lane 3=3:1-3; lane 4=5:1-1; lane 5=5:1-2; lane 6=5:1-3; lane 7=7:1-1; lane 8=7:1-2; lane 9=7:1-3; lane
10=20:1-1; lane 11=20:1-2; lane 12=100:1-1 and lane 13=100:1-2. Lower bands (shown inside a white box) of around 0.8 kb in
size (which is the estimated size of LASP1 fragment after cutting with AfeI and Not I HF) were obtained in each of the lanes
except lane 3. Thus, all of them appeared to be successful except for lane 3 that is ratio of 3:1-3.
1 2 3 4 5 6 7 8 9 10 11 12 13
31
The miniprep samples were then sent for sequencing after proper dilutions. Genewiz universal
primer CMV forward was used for sequencing. The results obtained seemed to be good
because the AfeI site was seen in most of the ratios except 3:1-1, 3:1-3, 5:1-1, 5:1-2 and 7:1-3
ratios. AfeI site was important as it was included in the plasmid map of LASP1-oligonuleotide to
favor blunt end ligation with ELP V96. The 7:1-2 and 20:1-2 ratios were then used in the next
ligation calculations.
3.2 Fusion of LASP1-oligonucleotide plasmid DNA with ELP V96
The restriction digest mixture of LASP1-oligonucleotide plasmid DNA was also analyzed on a
DNA gel after dephosphorylation similar to previous analysis done for the restriction digest
mixture. The sample was divided into two portions for loading on the gel.
Figure 11: DNA gel for restriction digest of LASP1-oligonucleotide plasmid DNA. The insert: vector ligation ratios 7:1-2 and 20:1-
2 from Table 12 were used for the restriction digest reaction. Two reactions per ratio were carried out so there were 4 reaction
tubes in all. Further the sample in each tube was divided into half to load on the gel. As a result, there were 8 bands obtained
on the gel. Lanes 1 and 2: tube 1 of 7:1-2 ratio; lanes 3 and 4: tube 2 of 7:1-2 ratio; lanes 5 and 6: tube 1 of 20:1-2 ratio; lanes 7
and 8: tube 2 of 20:1-2 ratio.
1 2 3 4 5 6 7 8
32
The DNA was extracted into 30 µl of EB buffer. NANODROP readings obtained were as follows:
Table 13: NANODROP readings for restriction digest of LASP1-oligonucleotide plasmid DNA.
Ligation ratio used for Restriction digest NANODROP readings (in ng/µl)
7:1-2 (1) 21.86
7:1-2 (1) 24.63
7:1-2 (2) 24.23
7:1-2 (2) 21.83
20:1-2 (1) 23.2
20:1-2 (1) 18.63
20:1-2 (2) 24.1
20:1-2 (2) 15.13
Restriction digest mixture of ELP V96 when analyzed on a DNA gel, a desired 1.4kb band was
obtained. This band was cut out, extracted and analyzed using NANODROP where the
concentration of DNA obtained was 14 ng/µl.
Figure 12: DNA gel for restriction digest of ELP V96. Panel A shows the 1.4 kb band obtained after loading the restriction digest
reaction of ELP V96 on the gel. Panel B shows the 1.4 kb band excised from the gel.
1.4 kb excised band
1.4 kb band
A B
33
When the ligation reaction of LASP1-oligonucleotide with ELP V96 was transformed using TOP
10 E. coli cells, only one colony was obtained for a 5:1 ratio (from table 9). The rest of the plates
showed no colonies at all.
Figure 13: Colony seen for 5:1 ratio of LASP1-oligonucleotide plasmid DNA and ELP V96 ligation reaction.
So, this single colony was picked, miniprep was performed and the NANODROP concentration
obtained for that miniprep was 204 ng/µl. The ligation product was confirmed through
sequencing. Maxiprep was then performed using Qiagen protocol and the concentration
obtained from NANODROP was 1040 ng/µl. This sample was then stored at -20 ℃ to be used
during the transfection procedure. The transfected cells were collected using
Radioimmunoprecipitation assay (RIPA) buffer. 10X RIPA buffer consisting of 25 mM Tris. HCl
pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS was used for the
procedure after dilution to 1X concentration with double distilled water. After collection of
transfected cells, the protein concentration was determined using BCA assay. The protein
concentrations obtained were 678 ng/µl, 720 ng/µl and 1105 ng/µl for wells number 4,5 and 6
of the 6-well plate, respectively, which were used for transfection. When western blotting was
34
performed to isolate the ligated product from transfected cells, there were no bands obtained
during imaging of the membrane. Both autofocus and manual exposure times were attempted
to obtain the images. But, upon imaging, only a grey spotted background was seen with hazy
bands in the beginning and no bands afterwards.
35
CHAPTER FOUR: DISCUSSION
Achieving the fusion of LASP1 with oligonucleotide posed many challenges. During the first
restriction digest reaction for LASP1 wild type plasmid, Antarctic phosphatase was added in the
same reaction as Hind III HF with an aim to optimize the steps. The total volume was adjusted
to 50 µl. Gel electrophoresis was performed and DNA was purified in case of LASP1 restriction
digest mixture while oligonucleotide mixture was used directly, that is without purifying it This
was because gel electrophoresis could not be performed for the oligonucleotide alone because
of its very small size of 28 bp. The reaction was then incubated for one hour at 37 ℃. But the
ligation reaction did not work, and there was no growth on the transformation plates. So, the
same steps were repeated with overnight digestion for both restriction digest reactions.
Positive and negative controls were maintained during transformation. There was little growth
on the positive control plate but still no growth on other plates. The growth on positive plates
assured that the LASP1 plasmid was in a good and workable condition but there was definitely
some problem with the restriction digest, dephosphorylation and/or ligation reactions. Further
trials were conducted by diluting restriction digest mixtures of plasmid and oligonucleotide,
increasing the amount of vector for ligation, conducting PCR purification for oligonucleotide in
place of gel electrophoresis. But still the ligation did not work. Possible reasons behind these
failures might be: insufficient buffer in the reaction and consequently less zinc required for
dephosphorylation; insufficient incubation the first time when two enzymes were used
together; and/or, incorrect quantities of functioning vector and inserts or lack of PCR
purification the first time. So, all these issues were addressed together, and the ligation was
then a success. The restriction digest was performed, the mixture was incubated for 1 hour and
36
heat inactivated. Then a sufficient amount of Antarctic phosphatase buffer containing zinc was
added and again incubated for 1 hour. PCR purification was performed for oligonucleotide,
proper quantities of vector and insert were used, and both positive and negative controls were
maintained for ligation. Finally, colonies were obtained for all the ligation ratios used. The
restriction digest for LASP1-oligonucleotide plasmid DNA was conducted using a small amount
of DNA (0.5 µg). But NANODROP concentrations obtained were low. So, the restriction digest
was repeated with a larger amount of DNA (2 µg). The NANODROP readings obtained were
higher when 2 µg of DNA was used as starting material were somewhat higher than previous
reactions. Moreover, the NANODROP concentration obtained after PCR purification of the
blunting reaction was higher than the amount of DNA used as starting material for the reaction.
The reason behind this might be the measurement of unreacted template or free dNTPS in the
mixture along with the desirable product. This might result in unsuccessful blunt end ligation of
LASP1-oligonucleotide plasmid DNA with that of the insert for ELP V96. In spite of this
presumed outcome, the higher reading was used for the ligation reaction to see if the reaction
works irrespective of the higher reading. But the reaction did not work out. There were no
colonies obtained on the plates when the ligation mixture was transformed into E. Coli cells. So,
all of the steps were repeated once again and the outcome was a single colony on
transformation plates for just one ratio of 5:1. This result is consistent with blunt-end ligations,
which are much more difficult to achieve, compared to sticky end ligations. That one colony
obtained was then used for all the further reactions. While performing the fusion of LASP1-
oligonucleotide with ELP V96, amino terminus of LASP1 was selected because this region as
explained earlier is involved in forming complexes with multimeric proteins and the plasmid
37
used in this project encoded for an N-terminal fusion protein with LASP1. In addition, other
successful ELP fusion proteins were created as N-terminal fusions. For example, it was reported
that the fusion of FK-506 binding protein 12 (FKBP) with amino terminus that is N-terminal of
ELP A192 causes reduction in rapamycin toxicity and also enables intravenous therapy in
xenograft model of breast cancer as well as in a murine autoimmune disease model
(Dhandhukia et al., 2017). Similarly, ICAM-1 binding peptide that is IBP was successfully fused to
N terminus of ELP A192 to create a fusion protein which was seen to internalize only into
Intracellular Adhesion Molecule 1 that is ICAM1 overexpressing cells thus confirming specificity
of IBP to ICAM1 of mouse (Ju et al., 2019). Moreover, enhanced green fluorescent protein (e
GFP) has been successfully fused to the N-terminus of LASP1, and this fusion protein appears to
behave as endogenous LASP1 (Okamoto et al., 2008). However, there are also LASP1 fusions
with the fluorescent protein fused to the C-terminus of LASP1, but we did not have the
opportunity to create and test these constructs. Western blot was performed to see if the
ligated product of LASP1-oligonucleotide with ELP V96 was expressed in the HEK293T cells. But,
the imaging for western blot was not successful. This result might be because: the protein was
not expressed in mammalian cells; there was incomplete transfer of cellular proteins onto the
membrane during western blotting; or there might be insufficient incubation of the membrane
into ECL mix causing the signal for target ligated product to be weak. Another reason behind no
bands appearing during western blot imaging is that the actin cytoskeleton is insoluble in RIPA
buffer. So, the structures associated with the actin cytoskeleton may not partition into the
supernatant fraction. It is possible to have missed any LASP1-ELP fusions binding to F-actin
38
structures; these may have fractionated in the pellet with RIPA solubilization and not the
supernatant, which was analyzed by western blotting.
39
CHAPTER FIVE: CONCLUSION
Cell motility is a central feature not only for embryonic development, immune cell function,
tissue repair and angiogenesis but also for pathological processes like cancer metastasis. The
actin cytoskeleton dynamics are known to power this cell migration through a large number of
actin-binding proteins having a role in its regulation (Fenteany et al., 2003). LASP1 is one such
protein which takes active part in cell migration and motility through localization in
lamellipodia, filopodia or focal adhesions (Schreiber et al., 1998) . It is also being studied as one
of the important targets for cancer treatment and as a tumor biomarker. Although there are
many available small molecule drugs that target the actin cytoskeleton directly, there is a
scarcity of specific inhibitors of actin-binding proteins and immediate factors involved in
regulation of actin dynamics and cell movement (Fenteany et al., 2003). Moreover, controlling
actin-based cell motility has received very less attention as a therapeutic approach as compared
to use of cell cycle regulators as a part of anticancer drug development (Fenteany et al., 2003).
Elastin like polypeptides and their fusion proteins can be a potential treatment option in this
area owing to their temperature dependent phase transition property which would enable
them to aggregate at desired intracellular places above their transition temperature. Through
this project, attempts were made to fuse LASP1 with ELP V96 using recombinant DNA
technology. ELP V96, which is an ELP with longer chain length, was used in initial fusion protein
construction to ensure that it transitions well at temperatures below physiological
temperature. The resulting ligation product was transfected in the HEK293T mammalian cell
line. Although expression in 293T cells was not successful as was evident from the western blot,
other cell lines could be transfected. The future steps in the project would include expression of
40
the fusion protein into Hela or HEK293 cell lines and confirming the expression through western
blots. Proper measures can be taken to optimize the western blot procedure if needed. Positive
controls like intact LASP1 plasmid and intact ELP V96 plasmid can be loaded into the wells for
western blot purposes. If the expression of fusion protein becomes successful, then the
expressed protein would be used to characterize the behavioral similarity between LASP1-ELP
V96 fusion protein and the endogenous LASP1. This would further open up avenues for
determining the effect on ELPs on LASP1 based actin motility and subsequent potential in
cancer treatment. The longer chain length of ELP V96 would favor its transition at lower
temperature of around 24 degrees. Thus, it would remain in aqueous solution at room
temperature but when injected into the cell (where the temperature is higher than room
temperature), it would convert into a coacervate thus acting like an artificial organelle. This
artificial organelle might then, through the presence of LASP1, activate actin-based motility.
One reason we can think of is pulling away of LASP1 from the focal adhesions or lamellipodia
into the interior of the cell in order to inhibit cell migration. The ELP after coacervation would
contract slightly thus causing a pulling action on LASP1 and bringing it towards the inside of the
cell. Movement of LASP1 from the peripheral structures into the interior of the cell due to
coacervate formation of LASP1-ELP fusion proteins has not been shown yet experimentally, but
it appears to be a plausible explanation for the effects caused by fusion of LASP1 and ELP V96.
This can also be one of the possible explanations supporting the utility of LASP1-ELP V96 fusion
protein in cancer treatment by targeting LASP1 based actin motility and inhibiting cell motility
and metastasis.
41
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Asset Metadata
Creator
Vaidya, Vishvesha
(author)
Core Title
Characterization of actin based motility in mammalian cells through LIM and SH3 domain protein 1 (LASP1) and elastin like polypeptide (ELP) fusion protein
School
School of Pharmacy
Degree
Master of Science
Degree Program
Pharmaceutical Sciences
Publication Date
04/24/2020
Defense Date
03/19/2020
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
actin-based motility,artificial organelle,ELP,extraction,fusion protein,gel electrophoresis,LASP1,ligation,mammalian cell culture,maxiprep,miniprep,nanodrop,OAI-PMH Harvest,pcr purification,restriction digest,temperature dependent phase transition property,transformation,western blotting
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Wang, Clay (
committee chair
), Haworth, Ian (
committee member
), Mackay, Andrew (
committee member
), Okamoto, Curtis (
committee member
)
Creator Email
vishveshavaidya@gmail.com,vvaidya@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c89-290900
Unique identifier
UC11673139
Identifier
etd-VaidyaVish-8317.pdf (filename),usctheses-c89-290900 (legacy record id)
Legacy Identifier
etd-VaidyaVish-8317.pdf
Dmrecord
290900
Document Type
Thesis
Rights
Vaidya, Vishvesha
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Access Conditions
The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...
Repository Name
University of Southern California Digital Library
Repository Location
USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA
Tags
actin-based motility
artificial organelle
ELP
extraction
fusion protein
gel electrophoresis
LASP1
ligation
mammalian cell culture
maxiprep
miniprep
nanodrop
pcr purification
restriction digest
temperature dependent phase transition property
transformation
western blotting