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Overexpression and interaction of Orb2 in S2 insect cells

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Overexpression and interaction of Orb2 in S2 insect cells

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Content OVEREXPRESSION AND INTERACTION OF ORB2 IN S2 INSECT
CELLS

by

Shruti Vijay Bendre

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfilment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR MEDICINE)

AUGUST 2020

Copyright 2020 Shruti Vijay Bendre
ii
Acknowledgements

It is my proud pleasure to express my feelings of gratitude to every person who has helped me
directly and indirectly towards working on this research project.

I express my deepest gratitude towards Dr. Ansgar Siemer for his untiring assistance,
encouragement, suggestions, sincere guidance and support.

I would like to thank my committee members, Dr. Ralf Langen and Dr. Ebrahim Zandi for their
guidance and scientific advice.

I would also like to thank Ms. Silvia Cervantes for carefully reviewing my thesis, for mentoring
me and being a strong support throughout the project. I would like to thank the former and current
Siemer lab members; Dr. Maria Soria, Connor Hurd, Kidist Ashami, Samriddhi Garg, Dilara Akca,
Dr. Alexander Falk and Rajashree Venkatraman for the most supportive lab environment.

I would like to thank the entire Department of Biochemistry and Molecular Medicine for giving
me the incredible opportunity of being a part of this huge family.

Last but not the least, I would like to thank my family and friends for their unceasing support and
love.

iii
TABLE OF CONTENTS

Acknowledgements………………………………………………...…………………………….ii
List of figures……………………………………………………………………………………..v
Abstract…………………………………………………..……………………….……......…….vi
Chapter 1: Introduction……………………………………………………...………………….1
1.1Amyloid fibrils………………………………………...………….………............1
1.2 Functional Amyloids……………………...…………………………...………….2
1.3 CPEB……………...……………………………………………......…………….4
1.4 Orb2 protein……….……………………………………...………...…………….4
1.5 S2 cells…………….………………………………...……………...…………….7
1.6 Interaction of Orb2 with calmodulin……........................................……………...9

Chapter 2: Results…….………………………………………………………………..……….11
2.11. Expression of Orb2A in S2 insect cells………………………….…...……….11
2.1.2 Purification of Orb2A320………………….………………..……..........…….17
2.2 Interaction of Orb2 with calmodulin……........................................…………….19

Chapter 3: Discussion…….……..………………………………………….………..……..….27
3.1 Expression of Orb2A320 in S2 insect cells………………………….………….27
3.2. Interaction of Orb2AEGFP with calmodulin by modulation of the intracellular
calcium levels………............................................................................................................…….29

Chapter 4: Materials and Methods…….…..…………………………….……….…..……….30
4.1 Initiating cell culture from frozen stock…..………………………….………….30
4.2 Passaging and maintenance of S2 cells…..…………………………….………..30
4.3 Transfection of cells…………………..…..……………………………….…….31
4.4 Transformation of cells (E.Coli) …..……………………………….…..……….31
4.5 Transfection……………………………………….…………..…..…………….31
a) Calcium phosphate transfection method…..………………………….….…….31
b) Effectene method…..………………………………………….………....…….32
4.6 Making a stable line……………………………………….…..…..…………….32
4.7 Cell lysis and expression assay………………………….………………...…….33
a) Using S2 cells……………………………..…….………………………….….33
b) Using E.Coli…..………...……………………....……………………………..34
4.8 Protein expression………………………………...……………………………..35
a) Using S2 cells……………………………..…….……………………………..35
b) Using E.Coli…..………...……………………....……………………………..35
4.9 Purification……..………………………………...……………………………...36
a) Using Ni-IMAC column………………….…….……………………………...38
iv
b) Using size exclusion column…………………………………………………..38
4.10 Western blot…………………………………...……………………………….38
4.11 DNA extractions (Mini & Maxi prep)………....………………………………39
4.12 Fixing cells for microscopy…………………...……..………………………...40
4.13 Plasmid design for Orb2A320……………….…….…………………………..40
References…………………………………………………………………………………...…..42

v
LIST OF FIGURES
Figure 1: Amyloid fibrils can be intracellular or extracellular, and functional or pathological…..3

Figure 2: Orb2A and Orb2B have a similar structure……………………………………………..5

Figure 3: S2 cells as observed under the light microscope using 100X lens……………………...8

Figure 4: Interaction of calcium with calmodulin………………………………………………..10

Figure 5: Schematic representation of the methods for transfection of S2 cells………………...12

Figure 6: Efficiency of transfection is the same for calcium phosphate and effectene method…13

Figure 7: Orb2A320 was successfully expressed in S2 cells……………………………...……..14

Figure 8: Longer incubation leads to increased protein expression……………………………...15

Figure 9: Orb2A320 purified successfully of Nickel column……………………………………17

Figure 10: Size exclusion purification shows a single peak suggesting pure elution……………18

Figure 11: Schematic representation of the workflow for testing the effect of intracellular
calcium modulation on Orbb2EGFP puncta……………………………………………………..19

Figure 12: Treatment of cells with 10μM calcium ionomycin was toxic for the cells…………..20

Figure 13: Imaging by fixing the cells on the slide using 4%PFA and Fluoromount-G
considerably improved the imaging quality……………………………………………………...21

Figure 14: Comparison of the effect of Ca Ionomycin and BAPTA-AM on Orb2AEGFP puncta
was difficult to determine due to low transfection efficiency……………………………………22

Figure 15: Transfecting cells using effectene gives a better transfection efficiency…………….23

Figure 16: Cells incubated for 48 hours and imaged by fixing show a clear, heterologous puncta,
which can be quantified and the effect of Ca ionomycin can be studied efficiently…………….23

Figure 18: Schematic representation of titration of ionomycin………………………………….25
Figure 19: Different concentrations of Ca ionomycin indicated that Ca ionomycin is toxic for the
cells………………………………………………………………………………………………26
Figure 20: Map of pMT/V5-His plasmid adopted from Invitrogen™…………………………...41
vi
Abstract
Amyloid refers to the fibrous, proteinaceous deposits found in organs and tissues. Often,
amyloids are associated with neurodegenerative diseases, however, there are also amyloids that
have many useful features and are non-pathogenic, referred to as functional amyloids. Orb2 is such
a functional amyloid. Orb2 is found in Drosophila melanogaster where it has been shown to be
important for long-term memory and regulating mRNA translation at the synapse. In the study of
long-term memory, it’s still not known how memories persist for years when the proteins involved
in the process are degraded within days.
Data suggests that Orb2 expressed forms amyloid fibrils and these may be responsible for
sustaining memory. Orb2 is composed of two isoforms, Orb2A and Orb2B. Here, we focus on
expression and purification of Orb2 from Drosophila Schneider (S2) insect cells. We have
optimized protocols for the efficiency of transfecting cells and have successfully made a stable
cell line expressing the protein. We are working towards purifying the protein from S2 cells using
chromatography techniques, and comparing this same protein to that expressed in E.Coli and
thereby better understand the effect of any post translational modifications that might have taken
place in the S2 cells.
Calmodulin (CaM) is present in very high concentrations in the CNS, up to 10-100 uM, and has
been shown to directly regulate several proteins involved in Long term memory (LTM). Previously
the Siemer lab has shown that Orb2A interacts with calmodulin in a calcium dependent manner,
which inhibits its aggregation in vitro. Here, we are testing the effects of intra cellular calcium on
the aggregation of Orb2 labelled with EGFP in S2 inset cells.
1
1. Introduction
1.1 Amyloid Fibrils:
Amyloid refers to the abnormal fibrous, extracellular, proteinaceous deposits found in
organs and tissues. Amyloid is insoluble and is structurally dominated by β-sheet structure.
3
They
are protein homopolymers that adopt diverse cross-β conformations. These non-branching fibrils
are stabilized via intermolecular contacts between β-strands, which align orthogonally to the fibril
axis to yield cross- β architecture.
4

Amyloids from different proteins share several structural properties: they all have a fibrillar
morphology and cross β structure, whereby intermolecular main-chain hydrogen bonding acts as
one major stabilizing interaction. Recent technological advances in structural biology, which
include solid-state nuclear magnetic resonance (SSNMR), and cryo-electron microscopy (cryo-
EM), have allowed for the determination of the structure of amyloids at the molecular level.
5

The fibrillar structure of amyloid fibrils is one that is used by many organisms, from bacteria to
humans, in order to achieve a diverse range of biological functions.
6
In some organisms, the unique
structural traits of the cross-β structural core have been exploited by nature to serve a multitude of
biological roles not merely restricted to the provision of structural framework. A number of
functional amyloids serving roles as diverse as surface protection and modification, mediation of
host interactions, pigment biosynthesis, hemostatic control, hormone storage and release and
signal transduction have been discovered.
6

Studying the mechanisms of formation of amyloid fibrils is of major importance since insights into
the mechanisms underlying polymerization of soluble, monomeric peptide into mature insoluble
2
fibrils may provide researchers with possible therapeutic approaches to halting, reversing or
avoiding amyloid formation.
3

1.2 Functional Amyloids
Amyloids were first identified in association with amyloidosis, human diseases in which
proteins and peptides misfold into amyloid fibrils. Subsequent studies have identified an array of
functional amyloid fibrils that perform physiological roles in humans. Given the potential for the
production of toxic species in amyloid assembly reactions, it is remarkable that cells can produce
these functional amyloids without suffering any obvious ill effects.
7

In humans, functional amyloids have been proposed to participate in an array of physiological
processes including pigmentation, the storage of peptide hormones, the fertilization of oocytes by
sperm, antimicrobial responses, regulated necrosis, and cellular response to stress.
7

3

Figure 1: Amyloid fibrils can be intracellular (c,d) or extracellular (a,b), and functional (a,c) or
pathological (b,d). a) Functional amyloid in biofilm production seen in organisms such as
E.Coli. b) Pathological amyloid plaques, typically seen in models of Alzheimer’s disease. c)
Functional Pmel17 scaffold protein involved in the production of melanin.
d) Pathological lewy bodies developed, typically seen in Parkinson’s disease.
Taken from (Knowles & Buehler, 2011)
6

4
1.3 CPEB
One of the most fascinating things about long term memory (LTM) is that it persists even when
the proteins involved in the mechanism are degraded. Synaptic plasticity and synaptic
potentiation are two of the critical factors which are responsible for providing the ability to
change overtime with response to stimuli. The cytoplasmic polyadenylation element binding
protein (CPEB) proteins are a family of sequence specific RNA-binding proteins and they
regulate the synaptic protein synthesis.
It has been observed that the CPEB homolog in Aplysia (ApCPEB), Drosophila (Orb2), and in
mouse (CPEB3) form amyloidogenic oligomers, which are critical for the persistence of long-term
synaptic facilitation and behavioral memory.
1
CPEBs belongs to a family of RNA-binding
proteins that both acts as a translational repressor and activator of its target mRNAs.
8

These observations let to a model for long – term memory in which external stimuli recruit a self-
sustaining amyloidogenic form of neuronal CPEB in the activated synapse, where CPEB then
maintains memory through the sustained and regulated synthesis of a specific set of synapse
proteins.
1

1.4 Orb2 protein
Drosophila has two CPEB family members. One of these, Orb plays a key role in the establishment
of polarity axes in the developing egg and early embryo but has no known somatic functions or
expression outside of the germline. Whereas, Orb2 mRNA and protein are found throughout
development in many different somatic tissues.
9
In the embryonic CNS, Orb2 appears to be
concentrated in cell bodies and mostly absent from the longitudinal and commissural axon tracts.
In contrast, in the adult brain, the protein is seen in axonal and dendritic terminals.
9

5

Orb2 is composed of two isoforms, Orb2A and Orb2B which differ only in their N-terminal
residues. Orb2A has a truncated N-terminus, with only the first 8 amino acids being unique to
Orb2A. Orb2B has an extended serine-rich N-terminus with over 150 amino acids preceding the
first common residue with Orb2A. Both Orb2A and Orb2B then have a glutamine/histidine-rich
region followed by a glycine-rich region, and finally two RRM RNA binding domains and a zinc
finger at the C-terminal end. The two isomers also differ in their biophysical properties; Orb2A
forms amyloid more efficiently than Orb2 both in vivo and in vitro.
1

Figure 2: Orb2A and Orb2B have a similar structure which includes an N terminus followed by
a glutamine rich region, a glycine rich region and then two RRM domain and a zinc finger
domain. However, they differ in their N terminus, where Orb2A has a much shorter N terminus
as compared to Orb2B which seems to be playing a key role in conferring the characteristic
properties to the fibril.
6
Orb2B is constitutively expressed in large quantities in a number of neurons, but Orb2A is
expressed ~100 fold less. Yet, deletion of Orb2A decreases the overall oligomerization of Orb2
suggesting that Orb2A is essential for the regulation of oligomerization.

7
1.5 S2 cells
The S2 cell line was derived from a primary culture of late stage (20-24 hours old) Drosophila
melanogaster embryos. Many features of the S2 cells suggests that it is derived from a
macrophage-like lineage.
S2 cells grow at 26° to 28°C without CO2 as a loose, semi-adherent monolayer in tissue culture
flasks and in suspension in spinners and shake flasks with a doubling time of about 18 to 24 hours.
They are easy to culture with a comparatively faster doubling time, can be grown at room
temperature without any specific CO2 requirements and can be scaled up to large volumes of
suspension culture.
Like previously mentioned, Orb2 is naturally expressed in Drosophila melanogaster and hence we
decided to express the protein in S2 insect cells as that would allow us to study the protein in its
native form with all the post translational modifications which is the focus of the first part of my
thesis.

8

Figure 3: S2 cells as observed under the light microscope using 100X lens. The cells are
typically round in shape and do not seem to be clustered.
9
1.6 Interaction of Orb2 with calmodulin

Calmodulin (CaM) (an abbreviation for calcium-modulated protein) is a multifunctional
intermediate calcium-binding messenger protein expressed in all eukaryotic cells.
Calcium/ calmodulin – dependent protein kinase II (CamKII) is a protein involved in the induction
of many forms of synaptic plasticity in the brain.
10
The calcium sensing protein calmodulin (CaM)
also interacts with amphipathic helices. Among its many functions in a large number of different
cell types, CaM is an important signal integration protein in neurons for long- term memory.
11

CaM is present in very high concentrations in the CNS, up to 10-100 uM
2
, and has been shown to
directly regulate several proteins involved in LTM.
CaM interacts with sequences that are both amphipathic and positively charged.
2
It is an
intracellular target of the secondary messenger Ca
2+
, and the binding of Ca
2+
is required for the
activation of calmodulin. The most common mode of CaM binding to these helices is dependent
on calcium. Ca
2+
binds to each of the four EF domains present in CaM. This creates a hydrophobic
domain in which many of the CaM’s target proteins interact.

10

As previously studied by a former member of our lab Dr. Maria Soria, we have hypothesized that
CaM might bind to the N-terminus of Orb2A in a calcium dependent manner and cause its
disaggregation. We have observed this effect in vitro and are currently trying to determine if we
see the same effect in vivo. (Section 2.2)

Figure 4: Interaction of Calcium with Calmodulin. Four calcium ions bind in the EF pockets of
calmodulin and the calcium – calmodulin complex then binds the alpha helices.
Made by Dr. Maria Soria.

11
2. Results
2.1.1. Expression of Orb2A320 in S2 insect cells
In this project, are aiming towards purifying the protein from S2 cells using chromatography
techniques, and comparing this same protein to that expressed in E.Coli and thereby better
understand the effect of any post translational modifications that might have taken place in the S2
cells.
The plasmid was transfected into S2 cells using two different transfection methods: The effectene
method and the calcium phosphate method. The calcium phosphate method was suggested by most
manuals including the Thermo Fisher Scientific™ manual for S2 cells. However, previous
experiments on modulating intracellular calcium levels suggested that transfection using effectene
provided better transfection efficiencies. Hence, the cells were transfected using both methods and
experiments were performed to determine which one would provide a better yield.

12

Figure 5: Schematic representation of the methods for transfection of S2 cells

13

After transfecting S2 cells using both methods, an expression assay was run to evaluate the
efficiency of each. Using traditional Coomassie stain, it was difficult to locate the band
corresponding to our protein of interest; hence a western blot using anti-His primary antibodies
was carried out.
The protein was expressed for 24 hours and 48 hours to determine the ideal time of expression
Figure 6: Efficiency of transfection is the same for calcium phosphate and effectene method.
a) SDS – PAGE analysis of protein expression in S2 cells 24-hour post – induction using calcium
– phosphate transfection.
b) SDS – PAGE analysis of protein in S2 cells 24-hour post induction using effectene method.

14

The western blot indicated that the protein was successfully expressed in S2 cells, using both
transfection methods. The difference in levels of protein expression, using the two different
methods, seemed insignificant. In addition, in both cases most of the protein appeared in the non-
soluble part of the cell lysate, which was not expected, as when we expressed Orb2A320 in E.Coli,
we always saw it in the soluble fraction of the cell lysate. The experiment was repeated using
longer protein expression times. Specifically, expressions of 2 and 4 days were used to determine
the ideal time of expression in order to maximize the yield.
Figure 7: Orb2A320 was successfully expressed in S2 cells
Western blot post cell lysis using anti – His primary antibody shows that the protein was
successfully expressed in S2 cells. A significant amount of the protein seems to be present in
the non-soluble part of the cell lysate.

15
Given that there was no obvious difference in the level of protein expression between the two
transfection methods, it was decided that the effectene method would be used to do all subsequent
transfections. The effectene methods requires just 2 days to transfect as compared to calcium
phosphate which takes 4 days and, in addition, the effectene method is also comparatively less
toxic to the cells.

Figure 8: Longer incubation leads to increased protein expression.
a) SDS-PAGE gel of expression analysis, 2 and 4-days post transfection
b) Western blot post cell lysis using anti – His primary antibody shows that the protein was
successfully expressed in S2 cells. A significant amount of the protein seemed to be present
in the non-soluble part of the cell lysate.

16
The cells were transiently transfected using effectene reagent and the protein was expressed for 2
and 4 days. The cells were then lysed, and an expression assay was performed. Again, the western
blot indicated that the protein was present in the non-soluble fraction of the cell lysate. Protein
expression for 4 days showed a stronger band which indicated more protein as compared to that
observed for 2 days.

17
2.1.2 Purification of Orb2A320

A stable line containing the plasmid of interest along with pCoBlast was developed and grown in
selective media containing blasticidin. Blasticidin is a potent translational inhibitor and acts as an
antibiotic, which allows us to select for cells that were successfully transfected, as they’ll have
resistance to blasticidin conferred by pCoBlast. After inducing expression in stably transfected
cells by adding copper sulphate which induces the metallothionein promoter, purification was
carried out. The soluble fraction of the cell lysate was loaded onto the nickel column after running
an expression gel.

The elution fractions collected from the Ni column elution were then loaded on to the size
exclusion column.

Figure 9: Orb2A320 purified successfully of Nickel column
Chromatogram of Nickel affinity purification showing a single sharp peak in the elution
indicating a pure protein but comparatively low yield (Peak 1).
Peak 1
18

The protein was eluted and appeared clean as observed on an SDS page gel, however the amount
of pure protein that was obtained was low. Interestingly, the protein also eluted of the size
exclusion column a lot earlier than it typically does when expressed in E. Coli. One explanation
for this is that the protein could have been aggregated and thus eluted earlier. As previously seen
in expression gels, a good amount of protein is present in the non-soluble fraction, and thus trying
to purify the protein from the non-soluble fraction might give higher yields.

Figure 10: Size exclusion purification shows a single peak suggesting pure elution
a) Chromatogram of size exclusion purification, showing a single peak suggesting a pure protein
elution but of low concentration which eluted much before it’s expected elution volume(44ml)
as observed in E.Coli (76ml).
b) SDS-PAGE analysis of peak 1b after elution from size exclusion column shows a light band
indicating pure protein present but with a comparatively low yield.
Peak 2
Peak 2
Orb2A320
positive
control
19
2.2. Interaction of Orb2AEGFP with Calmodulin
A former lab member, Dr. Maria Soria has shown that Orb2A interacts with calmodulin in a
calcium dependent manner, which inhibits its aggregation in vitro. To see if CaM has a similar
effect in cell culture, we are testing the effects of intra cellular calcium on Orb2 labelled with
EGFP in S2 inset cells.

Figure 11: Schematic representation of the workflow for testing the effect of intracellular
calcium modulation on Orb2AEGFP puncta
20
The cells were passaged about 10 times to make sure they were viable and healthy. Once the cells
reached the desirable density, they were transfected and then imaged after the incubation period.

Figure 12: Treatment of cells with 10µM calcium ionomycin was toxic for the cells
a) Cells transfected with Orb2AEGFP and incubated for 24hours show some puncta throughout
the slide.
b) Cells transfected with Orb2AEGFP and incubated for 24hours followed by treatment with 10
𝜇M Calcium ionomycin for 1 hour, the cells rounded up and typically lifted from the plate at
20x magnification.
21
Puncta was visible in some cells, however the imaging quality needed significant improvement.
The transfection efficiency observed was very low and the cells seemed to assume a rounded
morphology upon treatment with calcium ionomycin which ultimately seemed to be toxic to the
cells. Hence, it was difficult to draw conclusion on the effect of disaggregation of puncta and
troubleshooting experiments were carried out.

Figure 13: Imaging by fixing the cells on the slide using 4%PFA and Fluoromount-G
considerably improved the imaging quality.
(63X magnification)

63X magnification
22

Figure 14: Comparison of the effect of Ca Ionomycin and BAPTA-AM on Orb2AEGFP puncta
was difficult to determine due to low transfection efficiency.
a) Cells transfected with Orb2AEGFP and incubated for 48 hours followed by treatment with
2µM calcium ionomycin for 1 hour
b) Cells transfected with Orb2AEGFP and incubated for 48 hours followed by treatment with
100µM calcium ionomycin for 1 hour
63x magnification

23

Figure 15: Transfecting cells using effectene gives a better transfection efficiency
a) S2 cells transfected with Orb2AEGFP using effectene method, 24 hours post transfection
b) S2 cells transfected with Orb2AEGFP using effectene method, 48 hours post transfection,
with good transfection efficiency.

Figure 16: Cells incubated for 48 hours and imaged by fixing show a clear, heterologous puncta,
which can be quantified and the effect of Ca ionomycin can be studied efficiently.

24
Although the imaging quality was improved, the transfection efficiency was still low. Hence it was
difficult to determine the effect of calcium level modulation on the disaggregation of Orb2AEGFP
puncta.

The cells were fixed using 4%PFA and Fluoromount-G. Fixed cell imaging seemed to improve
the quality of images considerably when compared to live cell imaging. The puncta in cells was
clearly visible as seen in figure 9.

The transfection method was changed from calcium phosphate method to effectene method and
the transfection efficiency was greatly improved. The next challenge was to find the ideal time of
incubation for cells with ionomycin and the right concentration of ionomycin, such that the calcium
levels would be modulated, and thereby cause a clear effect on the Orb2AEGFP puncta, but which
would not be toxic to the cells. In an approach to determine this, a titration experiment was
performed with varying concentrations of ionomycin and varying incubation times.

25

Added ionomycin 24 hrs post transfection, incubated for 3 hrs after adding ionomycin
Added ionomycin 24 hrs post transfection, incubated for 24 hrs after adding ionomycin
Figure 18: Schematic representation of titration of ionomycin
26

When the cells were treated with 500nM calcium ionomycin for 24 hours, the effect was difficult
to comprehend. Whereas, when the concentration was increased to 1µM, most cells were dead and
autofluorescence was observed.

Figure 19: Different concentrations of Ca ionomycin indicated that Ca ionomycin is toxic for
the cells.
a) S2 cells transfected with Orb2AEGFP treated with 500 nM ionomcycin for 24 hours
b) S2 cells transfected with Orb2AEGFP treated with 1µM ionomcycin for 24 hours where most
cells were dead, and poor EGFP signal was observed due to autofluorescence indicating the
ionomycin was toxic to the cells.

—10 µm
27
3. Discussion
3.1 Expression of Orb2A320 in S2 insect cells

Here, we chose to express Orb2A320 in S2 insect cells because Orb2 is an endogenous protein of
Drosophila and S2 cells are derived from Drosophila embryos. The hypothesis behind this was
that expressing the protein in S2 cells would allow us to study the protein in its native state along
with all the post-translational modifications.

However, on expressing the protein in large quantities and purifying it, the yield was lower than
expected. Orb2A320 purified using S2 cells gave around 8 to 10 times lower yield when compared
to that from E.Coli. The protein also eluted much earlier (44ml) than that compared to protein
expressed in E.Coli (76ml). This observation could indicate that it aggregated inside the cells.
However, the change in elution profile could also be explained by an increase in the molecular
weight of the protein, which could be due to post-translational modifications which are
characteristic to S2 cells.

There has been evidence in literature
12
that the baculoviral system might be a better system to
express the protein in large volumes as compared to S2 cells. Baculoviruses are arthropod-specific
viruses that infect more than 600 host species.
13
An increase in the understanding of these viruses
led to the application of baculoviruses as an expression system.
13

The major restrains in using S2 cells for protein expression has been low transfection efficiency
14
. When S2 cells were transfected using the baculoviral system, almost 100% of the cells showed
28
gene expression and the cells also showed higher levels of protein expression. There has also been
a considerable use of Sf9 cells for manufacturing a large amount of viral and polyproteins.
15

Sf9 is an insect cellxs line from Spodoptera frugiperda which is a common cell substrate for
baculovirus-expressed biological products
15
especially because these cells have the capability to
recognize and process all the signal peptides, and process the post-translational modifications.
13

In future, we would like to try expressing Orb2A320 in insect cells using the baculoviral system,
which has been previously done to express full length monomeric Orb2
1
.

In future, we would like to try purifying the non-soluble fraction of the cell lysate as data from
the expression analysis showed that a significant part of the protein was present in that fraction.
We would also like to send the protein for mass spec analysis to compare the protein purified in
S2 cells with the one in E.Coli. We would also like to perform droplet formation studies on this
protein.

29
3.2 Interaction of Orb2AEGFP with calmodulin by modulation the intracellular calcium
levels
Previously, Orb2AEGFP puncta was visualized in S2 cells which was observed as puncta or
diffusely distributed indicating aggregation or no aggregation
15
. We decided to use the same
system to study the effect of modulating intracellular calcium levels using ionomycin as a promoter
of intracellular Ca
2+
, and BAPTA-AM as in intra cellular Ca
2+
chelator.

S2 cells have been previously used to study Natural Killer(NK) cell activation, by transfecting
them using Cellfectin reagent (Invitrogen™)
16
. There has also been evidence that S2 cells express
the protein more efficiently by transfection using lipofectin and electroporation. Where, lipofectin
was also used to create a stable cell line expressing GFP
17
. It would be interesting to try these
transfection methods and find out their effect, if any.
We hypothesized that the intracellular calmodulin levels are insufficient and overwhelmed by the
sudden increased calcium influx and we thought of troubleshooting this issue by over-expressing
calmodulin with Orb2AEGFP and then studying its effect on Orb2AEGFP puncta. So far, we have
been able to co-transfect the plasmid and are working on quantifying the puncta.

30
4. Materials and methods
4.1. Initiating cell culture from frozen stock.
Frozen S2 cell stock was purchased from ThermoFischer Scientific (Waltham, MA). Cells had
been stored in 10% DMSO were thawed in a water bath at 30C. All the contents in one vial
containing approximately 1x10^7 cells were transferred into a sterile tube containing
approximately 4ml of Schneider's medium. This was then centrifuged; the supernatant was
aseptically decanted, and the cells were resuspended in 5ml of fresh complete Schneider's medium.
The suspension was then transferred to a T-25ml flask and cells were incubated at room
temperature.
4.2. Passaging and maintenance of S2 cells
The cells were plated in complete Schneider’s media with penicillin and streptomycin and allowed
to grow until between 2x10
6
to 2x10
7
cells/ml. The concentration and viability were measured on
Orflo (Ketchum, ID) Moxi automated cell counter using propidium iodide. Viabilities over 98%
were typical.

31
4.3. Transfection of cells
The cells were transformed using the commercial Effectene kit purchased from Qiagen (Hilden,
Germany). The protocol was followed exactly as mention in the manual. 2ug of DNA was used
with 16ul of enhancer reagent with 50ul of effectene reagent per 35mm plate containing 5ml of
suspension in complete Schneider’s media. The cells were then plated in a 35mm plate and
incubated at room temperature as long as needed.

4.4. Transformation of cells (E coli)
Competent XL10 cells were taken from the refrigerator (-80°C) and thawed on ice. 100ul aliquot
was taken and approximately 1ul of DNA was added in a pre-cooled, sterile culture tube. The cell
incubated for 10 minutes and then subjected to heat shock by keeping in water bath at 42°C. The
culture was kept back on ice for 2 minutes and then 800ul of SOC medium was added. Next, the
culture was incubated at 37°C with shaking at 225 rpm (series 5, incubator shaker) for an hour and
then plated on pre warmed agarose plates containing penicillin. Plates were incubated overnight at
37°C and colonies were observed next day, which were either used the same day, or the plates
were stored at 4°C in the refrigerator.
4.5. Transfection
a) Calcium Phosphate transfection method:
S2 cells were grown to an adequate density and 3 x 10
6
cells in 3ml complete Schneider’s medium
with 100% FBS were seeded in a 35mm plate overnight. A mixture of 19ug recombinant DNA,
32
2mM CaCl2 and tissue culture sterile water referred to as Solution A was made. A second solution
containing 300uL 2X HEPES buffered saline was called Solution B. Solution A was slowly added
to solution B until solution A was depleted and mixed continuously to ensure fine precipitation
necessary for efficient transfection. This mixture was then incubated for approximately 30 to 40
minutes and a fine precipitate was formed; this was then added dropwise to the cells with
continuously swirling the plate after every drop. This mixture was then incubated for 16-24 hours
at room temperature. On the next day, cells were washed twice with Schneider’s complete medium
to remove the calcium phosphate solution and re-plated in the same plate in fresh, complete
Schneider’s medium and incubate till the desired time.
b) Effectene method:
The effectene transfection reagent was purchased from Qiagen™ and instructions in the manual
were followed exactly. 2ug of DNA was used per transfection and cells were transfected in a 60mm
sterile dish.
4.6. Making a stable cell line
Cells were transfected using the effectene method with 1ug of pMTV5/HisA/Orb2A320 DNA and
1ug of pCOBlast DNA (commercially purchased from Thermo Fischer). The cells were incubated
at room temperature for 2 days post transfection and then resuspended in Schneider’s medium
containing selective reagent (100mM blasticidin) and then incubated again for 2 weeks. The cells
were re-plated in new plates in selective medium and passaged when they reached the adequate
density. The cells were then passaged and maintained in selective medium.

33
4.7. Cell lysis and expression assay
a) Using S2 cells
Lysis Buffer
50mM Tris, pH 7.8
150mM NaCl
1% Nonidet P40 substitute
1mM PMSF
1 tablet of protease inhibitors per 10ml of lysis buffer solution

The cell suspension was spun down at 1000g for 10 minutes and the supernatant was decanted.
The cells were then resuspended in PBS and lysis buffer was added. 100ul of lysis buffer was
added per 1.5 ml of cell suspension which was vortexed for about 2 minutes, incubated for 1o
minutes on ice. This was then spun down at 1000g for 10 minutes and aliquots of the supernatant
(soluble fraction) and pellet (non-soluble fraction) were then taken to analyze for expression using
an SDS PAGE gel and western blot. 40ul of the aliquot was mixed with 20ul of SDS loading buffer
and heated at 95°C for 10 minutes. This sample was then loaded on a commercially available SDS
PAGE gel and allowed to run. The gel was stained with Coomassie blue dye solution and then de-
stained to visualize the protein bands.

34
b) Using E. Coli
Lysis Buffer
GT Buffer
25mM Imidazole
50ul of 10X DNAse 1 (Add fresh)
1mg/ml lysosome (Add fresh)

The cells were taken out from -80°C refrigerator and allowed to thaw on ice. 25ml of lysis buffer
was added to the cell pellet and the pellet was vortexed till it was completely dissolved in buffer.
The mixture was then sonicated using a cell disrupter sonicator™ (Heat systems model W-220F)
set at a power of 3 or 4 using 75% amplitude, 30 second pulse, and 1-minute cycle repeated for 5
rounds. The pellet mixture was kept cold throughout this procedure by keeping it on ice. The cell
lysate mixture was transferred in a centrifuge tube and spun down at 20,000 rpm at 4°C to separate
the soluble and non-soluble fractions using Sorvall® ss 34 rotor and the Sorvall® Evolution RC
centrifuge. The supernatant was further used for purification.

35
4.8. Protein expression
a) Using S2 Cells:
Stably transfected cells were split in selective medium once they reached the adequate density and
the protein expression was scaled up. The cells were induced using 100 mM Copper Sulphate and
allowed to express the protein for 48 hours.

b) Using E. Coli
pET28/Orb2A320 plasmid was transformed into E.Coli BL21 cells using the procedure metioned
above and the culture was plated on agarose plates selecting for kanamycin resistance. A single
colony was inoculated in LB broth the next day containing 25ul of Kanamycin (5mg/ml) and
incubated at 37°C 225rpm for about 4 hours. Once the suspension seemed cloudy, 5ml of the
suspension was expanded into 500ml of LB broth containing 500ul Kanamycin (5mg/ml) and
incubated at 37°C, 225 rpm the optical density was measure periodically until it reached between
0.6-0.7. The expression was then induced by adding 500ul isopropyl-1-thiol β D galactopyranoside
(1M) and incubated at 18°C for 20 hours. The cells were then spun down at 5000g for 15 minutes
at 4°C. The cell pellet was either stored at -80°C or purified.

36
4.9. Protein purification
The following buffers used:
1. GT Buffer (pH 7.6)
1M Urea
100mM KCl
10mM HEPES
0.1% Tween – 20
0.05% β mercaptoethanol (Add fresh)

2. Wash #1 for Ni column
GT Buffer pH 7.6
0.5% Trition X (Removes lipids)
0.05% β mercaptoethanol (Add fresh)

3. Wash #2 for Ni column
GT Buffer pH 7.6
0.5M NaCl (Removes Trition X and Nucleic Acids)
0.05% β mercaptoethanol (Add fresh)

37
4. Wash #3 for Ni column
GT Buffer pH 7.6
20mM Imidazole
0.05% β mercaptoethanol (Add fresh)

5. Wash #4 for Ni column
GT Buffer pH 7.6
1M NaCl (Prepares for 1M NaCl used for elution)

6. Elution buffer for Ni column
GT Buffer pH 7.6
1M NaCl (disfavors droplet/inclusion body formation)
500mM Imidazole
0.05% β mercaptoethanol (Add fresh)

Size exclusion buffer
GT Buffer pH 7.6

38
a) Using Ni-IMAC column
The protein was purified using a Ni-NTA IMAC affinity column (Sigma Aldrich™) and was pre-
equilibrated using 50ml of filtered, degassed water followed by 50ml of elution buffer and 50ml
of GT buffer at the flow rate of 5ml/minute. The sample to be loaded was filtered using an 0.45µm
corning filter and the FPLC sample line was placed in the filtered sample. The sample was then
loaded on to the FPLC at the rate of 2.5 ml/min. The column was washed using wash #1, #2, #3,
#4 respectively and the protein was eluted using 50ml of the elution buffer. The fractions that
contain the protein of interest were then combined by selecting them using the chromatogram.
b) Using size exclusion column.
The Hi Prep 16/60 sephacryl S 300 SR column by GE Healthcare™ was used. The column was
cleaned using 2 Column Volumes (CV) of degassed, filtered distilled water, 0.25 CV of NaOH
followed by 2 CV of degassed, filtered distilled water again. This column was then equilibrated
using 2 CV BT buffer. The sample collected after Ni column purification was loaded at the flow
rate of 0.5ml/min followed by 1 CV of GT buffer for elution. The protein was collected and flash
frozen using liquid nitrogen for further analysis.

4.10. Western blot
SDS page was using the normal protocol for SDS-PAGE using a reference ladder. 1liter 10X
transfer buffer was prepared by mixing 30.3 g Tris, 144.1 g Glycine which was then diluted to 1X
using 100ml of 10X transfer buffer, 200ml methanol and 700 ml water. Membrane transfer was
39
then done using the 1X transfer buffer at 100mV for 1 hour. The membrane was soaked in blocking
solution (5% w/v milk powder) followed by washing thrice with TBST buffer (8g NaCl, 2.4g Tris,
10µl Tween 20 in 1liter water, pH = 7.6). The membrane was then immunostained with primary
antibody solution (Anti- 6XHis epitope tag, rabbit IgG antibody) by either incubating for 1 hour
at room temperature of incubating overnight at 4°C with constant agitation. The membrane was
again washed thrice using TBST buffer and the same step was repeated for secondary antibody
(Anti Rabbit -IgG, goat peroxidase conjugated antibody). After poring off the secondary antibody
and washing the membrane thrice with TBST, the membrane was soaked in 2ml of GE detection
solution 1 and GE detection solution 2 for 30 seconds and developed in ____.

4.11. DNA extractions (Mini & Maxi prep)
XL10 gold cells were transformed by the protocol mentioned above, a single colony was picked
the next day and inoculated in a 500ml flask containing 20ml sterile LB media with ampicillin.
The culture was incubated at 37°C with shaking at 225 rpm for about 20 hours. The suspension
was then spun down at 5000 rpm for 20 minutes at 4°C. DNA was then isolated using the Zippy™
plasmid miniprep or maxiprep kit and following the Zippy™ instruction manual. The DNA was
measured using nanodrop ND- 1000 spectrophotometer and sent out for sequencing with Genewiz,
Inc. to confirm the DNA sequence.

40
4.12. Fixing cells for microscopy
After transfection, S2 cells were incubated for the desired amount of time at room temperature.
1ml – 2ml of cell suspension was used per slide. The cell suspension was spun down at 3000g for
5 minutes. The cell pellet was then resuspended in 250ul Phosphate Buffered Saline and 20ul 4%
Paraformaldehyde, to a final concentration of 2%. The suspension was incubated for 10 minutes
at room temperature and the supernatant was removed carefully without disturbing the pellet. 40ul
of Fluoromount-G was then added to the cell pellet and mixed carefully avoiding air bubbles. The
resuspended cell solution was then transferred to a microscopy slide and the coverslip was applied.
The excess media was wiped off from the sides of the slide and then imaged.

4.13. Plasmid design for Orb2A320
A plasmid was designed using the S2 cell vector which is pMTV5/HisA containing the Orb2A320
sequence. It is an inducible plasmid with a metallothionein promoter. It is comparatively small in
size (3.5kb). We cleaved the plasmid using the restriction enzymes EcoRI and Age1, which
resulted in the cleavage of the V5 epitope. The plasmid has a 6XHis tag which makes it easy to
purify the protein using a Nickel chelating resin and perform western blot to detect the protein.
41

Figure 20: Map of pMT/V5-His plasmid adopted from Invitrogen™. The plasmid has a
drosophila metallothionein (MT) promoter which allows inducible expression of heterologous
proteins. It also has a bla promoter and bla resistance gene which permits selection of
trasnformants by using blasticidin.
42
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Abstract (if available)

Abstract Amyloid refers to the fibrous, proteinaceous deposits found in organs and tissues. Often, amyloids are associated with neurodegenerative diseases, however, there are also amyloids that have many useful features and are non-pathogenic, referred to as functional amyloids. Orb2 is such a functional amyloid. Orb2 is found in Drosophila melanogaster where it has been shown to be important for long-term memory and regulating mRNA translation at the synapse. In the study of long-term memory, it’s still not known how memories persist for years when the proteins involved in the process are degraded within days. ❧ Data suggests that Orb2 expressed forms amyloid fibrils and these may be responsible for sustaining memory. Orb2 is composed of two isoforms, Orb2A and Orb2B. Here, we focus on expression and purification of Orb2 from Drosophila Schneider (S2) insect cells. We have optimized protocols for the efficiency of transfecting cells and have successfully made a stable cell line expressing the protein. We are working towards purifying the protein from S2 cells using chromatography techniques, and comparing this same protein to that expressed in E.Coli and thereby better understand the effect of any post translational modifications that might have taken place in the S2 cells. ❧ Calmodulin (CaM) is present in very high concentrations in the CNS, up to 10-100 uM, and has been shown to directly regulate several proteins involved in Long term memory (LTM). Previously the Siemer lab has shown that Orb2A interacts with calmodulin in a calcium dependent manner, which inhibits its aggregation in vitro. Here, we are testing the effects of intra cellular calcium on the aggregation of Orb2 labelled with EGFP in S2 inset cells.

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Asset Metadata

Creator Bendre, Shruti Vijay (author)

Core Title Overexpression and interaction of Orb2 in S2 insect cells

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Medicine

Publication Date 07/21/2020

Defense Date 05/06/2020

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

Tag amyloid protein,long term memory formation,OAI-PMH Harvest,Orb2

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Siemer, Ansgar (committee chair), Langen, Ralf (committee member), Zandi, Ebrahim (committee member)

Creator Email sbendre@usc.edu,shrutibendre3@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-337690

Unique identifier UC11663771

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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...

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