Study of integrin αIIbβ3 transmembrane and cytosolic domain interaction and the expression of extracellular domain by Pichia Pastoris

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Content Study of integrin αIIbβ3 transmembrane and cytosolic domain interaction and the expression of
extracellular domain by Pichia Pastoris
by
Jiaqi Xiao
A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
MEDICAL PHYSIOLOGY
August 2021
Copyright 2021 Jiaqi Xiao

ii

ACKNOWLEDGEMENTS
Time goes by so fast that I barely notice it, the master life has been past. It is the first step
in my future and a big step in independence. Looking back on these two years, I have had
uncertainty about my studies, the unknown and awe of research, and now I am more aware of the
sacredness of scientific research as I graduate.
During the two years, I was bathed in the good learning atmosphere of medical physiology
department. I received the guidance of Prof. Tobias Ulmer, a graduate tutor of the Department of
Medical Physiology. My research ability has been greatly improved during the year. From the
beginning of the thesis to the end of the topic, it is inseparable from the teacher's careful guidance.
The research attitude of Tobias Ulmer and the constant teachings have become valuable
experiences that have influenced my life.
I have also been grateful to my parents for being far away from home but still encouraging
me daily and giving me full support in terms of money and spirit. I am grateful to my best friend
for my two years of companionship and have helped me greatly in my studies and life. I really
appreciated Dr. Harvey Kaslow for offering a lot of help in my study life and suggestions when I
felt lost. I would love to express my gratitude to my great committee members Dr. Ansgar Siemer
and Dr. Ralf Langen for giving me good advice and attending my defense. I would also like to
thank the lab members: Alan Situ, Han Vu, Xuhang Dai and Zhai Dai for their guidance and help
during the initial research period. I am grateful to my idol Ten for saving me when I was frustrated.
I also sincerely thank the experts and professors who reviewed the papers in time, thank you for
your valuable suggestions on my thesis!

iii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS………………...…………………………………………………...ii
LIST OF TABLES…………………………………………………………………………….v
LIST OF FIGURES...………………………………………………………………………...vi
ABBREVIATIONS………………...…………………………………………………………..viii
ABSTRACT……………………………………………………………………………………ix
CHAPTER 1: INTRODUCTION….……………………………………………………1
1.1 Integrin related disease….…………………………………………………………………….1
1.2 Integrin structure and function….…………………………………………………………......2
1.3 Integrin αIIbβ3….…………………………………………………………..…………………5
1.4 Protein Purification….…………………………………………………………..…………….6
1.4.1 Immobilized Metal Ion Affinity chromatography…………………………………7
1.4.2 Fast Protein Liquid Chromatography……………………………………………...7
1.5 Pichia pastoris expression system……………………………………………………………..8
1.6 Nuclear Magnetic Resonance…………………………………………………………………9
CHAPTER 2: MATERIALS AND METHODS.……………………..…………….………..11
2.1 Gene construct………………………………………………………………………………..11
2.2 Overexpression of fusion protein-PET44-His6-TEV-xxx constructs………………………..14
2.3 Bicelles mimics test………………………………………………………………………….18
2.4 Transformation of Construct DNA into Pichia Pastoris……………………………………...19
2.5 Expression test of construct………………………………………………………………….21
2.6 Western blot………………………………………………………………………………….21
CHAPTER 3: RESULTS……………...…………………………………………………….….24
iv
3.1 Interaction of the cytosolic and transmembrane domain between αIIb and β3……………...24
3.2 αIIbβ3 extracellular domain expression……………………………………………………...29
3.3 αIIb and αV Thigh-calf1-calf2 domain expression…………………………………………..36
CHAPTER 4: DISCUSSION………………………………………………..………….……38
REFERENCES………………………………………………………………..………………..41

v

LIST OF TABLES
Table 1: NMR titration group for αIIbβ3 cytosolic and transmembrane domain………………..27

Table 2: Primer design of αIIb (L1-P452)……………………………………………………….31
Table 3: Primer design of β3(V58-D434) / β3(D109-K354)…………………………………….32
Table 4: Primer design of αIIb leg domain………………………………………………………36
Table 5: Primer design of αV leg domain………………………………………………………..37

vi
LIST OF FIGURES
Figure 1: Integrin protein family…………………………………………………………………..1
Figure 2: General structure of integrin…………………………………………………………….3
Figure 3: Integrin states. a. Inactive state b. Active extended state. c. Active clustered state…….4
Figure 4: ÄKTA prime system……………………………………………………………………8
Figure 5: Pichia Pastoris…………………………………………………………………………..8
Figure 6: NMR machine…………………………………………………………………………..9
Figure 7: Illustration of fusion proteins used for αIIb (Ala963Cys) and β3 (Gly690Cys)
transmembrane and cytosolic peptides…………………………………………………………..24
Figure 8: SDS-PAGE results of different bicelles combination. 1. CHAPSO/DMPC/DMPG 2.
CHAPS/DMPC 3. CHAPS/DMPG 4. DHPC/POPC 5. CHAPSO/DMPC 6. CHAPS/POPC…...25
Figure 9: Relative quantities of accumulated αIIbβ3 cytosolic and transmembrane species as a
function of membrane mimic…………………………………………………………………….26
Figure 10: Chemical Exchange in NMR. k: reaction rates. Δf: chemical shift time scale………28
Figure 11: Crystal structure of the chosen extracellular domain (green). a. αIIb (L1-P452) b.
β3(V58-D434) c. β3(D109-K354)……………………………………………………………….29
Figure 12: Plasmid Map of pPICZα……………………………………………………………...30
Figure 13: Plasmid Map of pPIC9k……………………………………………………………...30
Figure 14: Western blot results. a. pPic9k-alpha-mating-KREAEA-FLAG-CD33(D140-T232)
C169S-His6-TEV-aIIb (L1-P452) b. pPic9k-alpha-mating-KREAEA-FLAG-CD33(D140-T232)
C169S-His6-TEV-b3(D109-K354). c. pPic9k-alpha-mating-KREAEA-FLAG-CD33(D140-
T232) C169S-His6-TEV-b3(V58-D434)………………………………………………………...34
vii
Figure 15: Western blot results. a. pPIC9K-alpha-mating-β3(D109-K354)-FLAG-His6 b.
pPIC9K-alpha-mating-β3(V58-D434). c. pPIC9K-alpha-mating-β3(V58-D434) with reducing
agent……………………………………………………………………………………………...35
Figure 16: Crystal structure of designed construct. a. αIIb red-thigh/yellow-calf1/blue calf2 b. αV
blue-thigh/yellow-calf1/grey-calf2………………………………………………………………36
Figure 17. Western blot result. αIIb thigh-calf1-calf2…………………………………………...38

viii
ABBREVIATIONS
DHPC (1,2-dihexanoyl-sn-glycero-3-phosphocholine)
POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol)
CHAPS (3-[(cholamidopropyl) dimethyl-ammonio]-1-propane sulfonate)
DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine)
CHAPSO (3-([3-cholamidopropyl] dimethylammonio)-2-hydroxy-1-pro-panesulfonate)
DMPG (1,2-dimyristoyl-sn-glycero-3-phosphoglycerol)
SDS (sodium dodecyl sulfate)
PAGE (Polyacrylamide gel electrophoresis)
NMR (nuclear magnetic resonance)
Cryo-EM (cryo -electron microscopy)
E. coli (Escherichia coli)
RGD (Arginylglycylaspartic acid)
rcf (relative centrifugal force)
rpm (revolutions per minute)
hr (hour)

ix
ABSTRACT

Integrin is a family of heterodimeric protein which has small cytosolic, transmembrane, and
large extracellular domain and could act through two mechanisms: inside-out and outside-in
pathway. Upon agonist stimulation, the transduction of inside-out signals leads integrin αIIbβ3 to
switch from a low- to high-affinity state for ligands, which is associated the structure change of
cytosolic and transmembrane domain. NMR analysis is the best way to study the αIIbβ3 complex
structure change in binding. Apart from inside-out signaling, ligand binding can reverse promote
outside-in signaling and drive essential platelet function, which is related to the structure of αIIbβ3
extracellular domain. The crystal structure of αIIbβ3 extracellular domain has been determined,
however, it has never been studied in solution formation. Due to its large size, Pichia Pastoris yeast
expression system is employed to synthesize the extracellular domain instead of E. coli system
used in cytosolic and transmembrane domain. The construction of integrin αIIbβ3 model will
precisely and accurately explain the role of integrin in transmitting extracellular signal into
recipient cells and exporting cellular regulations to external environment in blood hemostasis and
thrombosis. The reveal of the real structure of integrin αIIbβ3 could also boost the development of
new drugs and provide solid experiment foundation for further structure determination with
different integrin family proteins.

1

CHATPER 1: INTRODUCTION
1.1 Integrin related disease
Integrins are a widespread and diverse family of proteins that is functional in various cellular
biological processes. Integrins are able to play a role in many different diseases. For example,
cardiovascular, autoimmune, metabolic diseases, fibrosis, and cancer are all related to integrins.
Integrins are heterodimers that include an α subunit and a β subunit. The major function of
integrins is mediating many cell-cell and cell-extracellular matrix interactions. There are 18 α
subunits and 8 β subunits have been found until now. The combination of α and β can constitute
24 kinds of integrins in total. Different integrin can recognize and combine with different
components including extracellular matrix components such as fibrinogen, collagen, laminin,
firbronection and vitronectin, integrin-binding sites on cell surface such as arginine-glycine-
aspartic acid (RGD) sequence and others(1).

Figure1: Integrin protein family(2).

2
Among all the integrin family proteins, some subfamilies such as α4 and β2 subfamilies are
capable of controlling the interactions that are related to the migration of immune cells from the
general circulation to the sites of inflammation. In addition, integrins facilitate the retention of
immune cells at sites of chronic inflammation by binding to the extracellular matrix components(3).
Fibrosis is important in many chronic diseases and includes a pathological process of
formation of excessive extracellular matrix. The pro-fibrotic cytokine TGF-β is a key regulator of
fibrosis(4). Several integrins of the RGD-binding group play a dominant role in local activation of
TGF-β in diseased tissues. Inhibition of these integrins is therefore a promising strategy for treating
chronic fibrotic diseases.
TGF-β is also a key target for cancer treatment. TGF-β related pathway impacts both immune
and non-immune cell types which are used to to support tumor growth and reduce the potential of
anti-tumor immune responses(5). Therefore, inhibition of integrins which can activate TGF-β
offers as a powerful therapeutic approach in oncology.
1.2 Integrin structure and function
All the integrin’s function is related to its conformation. Integrins are αβ heterodimeric
receptors that mediate divalent cation-dependent cell-cell and cell-matrix adhesion through
tightly regulated interactions with ligands(6).
Integrin structure is usually composed of a large extracellular domain, and small
transmembrane and cytosolic tail. The short intracellular cytoplasmic domains may associate
directly with numerous cytoskeletal proteins and intracellular signaling molecules. These
associated proteins provide a basis for modulating fundamental cell processes and various
biological outcomes including proliferation, migration, cell differentiation, and apoptosis by
regulating signal transduction pathways(7), which can be called as inside-out signaling pathway.
3
In reverse, the large extracellular domain is responsible for the outside-in signaling pathway. When
interacted with ligands, the binding can generate formation and remodeling of focal adhesions,
which change cell shape, gene expression, and tissue organization.

Figure2: General structure of integrin(8).

The large extracellular domain of integrin α subunit usually includes I-domain, β-Propeller,
Thigh, Calf-1, and Calf-2. α1, α2, α10, α11, αD, αL, αE, αM, αX contain the I-domain structure,
which is crucial for ligand binding sites. α3, α4, α5, α6, α7, α8, α9, αV, αIIb contain no I-domain
but constitute the ligand binding sites by β-Propeller. Integrin β subunits have the I-like domain
which is crucial for ligand binding as well, and a PSI domain, a hybrid domain, four epidermal
growth factor (EGF) 1-4, and a β tail.

4

Figure 3: Integrin states(9). a. Inactive state b. Active extended state. c. Active clustered state.
The structure of integrin is highly flexible and can be adjusted based on its binding states.
In the inactive state, integrins are in a bent conformation and the transmembrane and cytoplasmic
regions are closely associated. Integrins can be activated by talins or kindlins, then the cytoplasmic
and transmembrane domain will separate, as well as the extracellular domain become extended.
Extracellular binding could happen in this active-extended conformation. After the ligand binding
occurs, several integrin subunits will cluster at the membrane, which is necessary to send
intracellular signals to form focal adhesions and actin cytoskeletal assembly.
However, even the general state change model has been studied, there are some problems
remain. A Cryo EM study before revealed a less compact conformation of αIIbβ3 with a different
arrangement of leg domains(10). Furthermore, two other studies have also reported extended
conformations of αIIbβ3 but failed to find a bent conformation(11, 12). Therefore, integrin αIIbβ3
is one of the most worth studying subunit of integrin family.

5
1.3 Integrin αIIbβ3
Integrin αIIbβ3 is the most abundant receptor on platelets, which can bind to fibrinogen
and von Willebrand factor to mediate platelet aggregation. Inherited mutations of either αIIb or β3
could result in the bleeding disorder called Glanzmann’s thrombasthenia. Antagonists of αIIbβ3 is
always prescribed for the prevention of thrombosis(13, 14).
As mentioned above, the most important character of the inactive state of integrin is their
interaction between of the transmembrane and cytosolic domain. The current best model of the
complex is coming from computational calculations with experimental restraints, which suggests
that the state chance from inactive to active-extended by the talin or kindlin is achieved by the
disconnection of the αIIbβ3. However, atomic-level structure dynamics of the αIIbβ3 interaction
are still required for the inactive state. Interaction between only the transmembrane domains has
been visualized using cryo-microscopy and single particle reconstruction of detergent-solubilized
intact αIIbβ3 integrins. As the only one that can study the interaction dynamic technique, NMR
spectroscopy is highly suitable to contribute to the structural and dynamic characterization of
transmembrane and cytosolic complex of αIIbβ3. Therefore, in our study, a series of NMR titration
experiments have been set to test their interaction.
Apart from the inactive state, the active state transition is also a difficult problem. Usually
upon extension, the head domain of integrin can remain in the closed conformation. But the bent
state can transit to an open conformation with high affinity for ligand as shown in crystals of the
αIIbβ3 headpiece(14). However, there is currently no known feature of integrin structure that
would enable cytoskeleton binding to couple to the extended, open conformation with high affinity
for ligand. This would appear to be important to fulfill the key role of integrins in integrating the
extracellular and intracellular environments. To study the ligand binding dynamics of integrin
6
αIIbβ3, a complete single head domain of αIIbβ3 need to be expressed first. In our research,
expression of different subdomains of head domain has been tested.
Except for the head domain expression, the leg domain of integrin αIIbβ3 is also an
important intermediate transmission structure of integrin state transition. More importantly, a
previous Cryo-EM study on αIIbβ3 revealed a less compact conformation with a different
arrangement of leg domains. Furthermore, there also has been reported that the overall
arrangement of α domains in the αIIbβ3 is similar to that in αVβ3crystals. Those two closely related
integrins, αIIbβ3 and αVβ3, share high structural homology and even bind to similar ligands in an
RGD-dependent manner. The high similarity gives us insight to the comparison study of the leg
domain of αIIb and αV. Previous research has shown that despite the similarity of the function and
structure of those two subunits, their response to ligand binding is so different. αVβ3 showed a
strong activation event in the presence of Mn2+, while the αIIbβ3 only showed a limited
activation(15).
Research of switching the α leg domain of those two subunits has revealed that the leg
domain is responsible for the ligand binding difference. To further study the character of leg
domain conformation of αIIbβ3 and αVβ3 and their influence on the ligand binding difference
with NMR, expression experiments of αIIbβ3 and αVβ3 have been conducted.
1.4 Protein Purification
Proteins are always obtained from a tissue or by their overexpression in an expression
system such as bacteria, yeast, or mammalian cell. Protein purification is the most important and
beginning step of harvesting the target protein. Protein purification is isolating proteins based on
their physical properties’ differences.

7
1.4.1 Immobilized metal ion affinity chromatography
Immobilized-metal affinity chromatography (IMAC) is a widely used technique for
purifying recombinant proteins with specific affinity tag. IMAC can be conducted under
denaturing or nondenaturing condition. The interaction is working through the interaction between
multiple electron donors of the specific affinity tag with a transition metal ion including Co2+,
Ni2+, Cu2+, Zn2+ chelated to a solid-phase column. The affinity tag used in our lab is mostly
polyhistidine, ranging 6–12 residues in length tagged to the N- or C-terminus of our target protein
sequence. Among different length of polyhistidine, 6-His is the most common and the histidine
imidazole ring can work as the electron donor. (16).
1.4.2 Fast Protein Liquid Chromatography

Fast protein liquid chromatography (FPLC) is a technique that belong to liquid
chromatography and is often used to purify protein mixtures. Chromatography separation principle
is usually composed of a mobile phase and a stationary phase. Not like some other forms of
chromatography, separation is completed due to the different components of a mixture have
different affinities for two materials. In FPLC the mobile phase is called as a “buffer”. For IMAC
system, the buffer always has different concentrations of imidazole to compete with the
polyhistindine for biding to the metal-charged resin and thus is used for elution of the protein from
an IMAC column. Typically, a low concentration of imidazole is added to both binding and wash
buffers to interfere with the weak binding of other proteins and to elute any proteins that weakly
bind. Polyhistidine tagged protein is then eluted with a higher concentration of imidazole(17).
8

Figure 4: ÄKTA prime system.
ÄKTA prime system is a flexible chromatography system that is widely used in the process
of FPLC. The buffer flow rate can be controlled by a positive-displacement pump and is normally
kept constant. The composition of buffer can be adjusted and varied by taking fluids in different
proportions from external reservoirs. The stationary phase is a resin that composed of cross-linked
agarose beads packed into a cylindrical glass or plastic column. FPLC resins are available in a
wide range of bead sizes and surface ligands depending on the application.
1.5 Pichia Pastoris expression system

Figure 5: Pichia Pastoris.
9
Pichia pastoris is a methylotrophic yeast which is highly successful used for expression of
heterologous proteins. In standard research, the bacterium E. coli is the most frequently used
organism for expression system. E. coli has the characters such as fast growth rate, simple growth
conditions. The biggest advantage of Pichia compared to E. coli is that Pichia is capable of
forming disulfide bonds and glycosylation in proteins(18). Besides, several factors have
contributed to its rapid acceptance. First, a promoter derived from the alcohol oxidase I gene
(AOX1) of pichia pastoris is perfect for the controlled expression of foreign genes. Secondly,
pichia pastoris allows appropriate folding and secretion of recombinant proteins to the external
environment of the cell. Moreover, the expression of endogenous secretory proteins in the pichia
pastoris system is limited which makes the purification of recombinant protein very easy(19).
In our study, pichia pastoris was used as the main system to express the large head and leg
domain of integrin.
1.6 Nuclear Magnetic Resonance (NMR)

Figure 6: NMR machine.

Currently there are three main research techniques for structural biology: single crystal X-
ray diffraction (SC-XRD), nuclear magnetic resonance (NMR) and cryo-electron microscopy
10
(Cryo-EM). However, there is no universal method since all three of them offer unique advantages
as well as limitations. In my research, NMR was employed as the main type of technology of
studying protein structure. Nuclei are charged, fast spinning particles. The nucleus can interact
with the surrounding atoms. Therefore, through nuclear magnetic resonance spectroscopy,
structural information of a given molecule can be obtained. When studying protein structure, its
secondary structure, such as α-helix and β-sheet can reflect the different arrangement of atoms.
The spacing of the atomic nuclei in different secondary domains, the interaction between nuclei,
and the dynamic characteristics of polypeptide segments can all directly reflect the three-
dimensional structure of proteins(20). All of which can contribute to spectroscopic behaviors of
the analyzed sample, thus providing characteristic NMR signals.
The most important advantage of the NMR method is that the three-dimensional structure
of macromolecules in the natural state can be measured directly in solution, and NMR could
provide information about dynamics and intermolecular interactions, which can’t be achieved by
X-ray crystallography and Cryo-EM.

11
CHAPTER 2: MATERIALS AND METHODS
2.1 Gene construct
2.1.1 Prepare vector
(1). Transform XL10 cells with vector and plate. Use a single colony to inoculate 4.5 ml of
LB medium containing the appropriate antibiotic(s) in 14 ml Round Bottom Tube w/ Snap
Cap tube and grow o/n at 37 °C, 225 rpm.
(2). Load 2 ml of o/n culture in 2 ml tube and spin down (2 min, 2500g). Repeat once more,
then extract plasmid DNA from cell pellet using the QIAGEN Miniprep kit (cat. no. 27106).
Elute DNA using 46 μl buffer EB.
(3). Take 43 μl of vector DNA into new tube, add 5 μl 10X restriction enzyme buffer, 1 μl
restriction enzyme I, 1 μl restriction enzyme II, 0.5 μl 100X BSA. Mix by pipetting up and
down, then incubate at 37 °C for 2 hr.
(4). Prepare 30 ml of 1.0% agarose gel in 0.5X TBE buffer. Heat gel in microwave until all
agarose has dissolved, then stir and heat more. Replenish evaporated water with MilliQ-
H2O. To slightly cooled agarose (<60 °C) adds ethidium bromide to 0.5 μg/ml (in the
hood), then pour the gel. Wait for at least 30 min to cool, then fill gel tank with new 0.5X
TBE buffer; fill to 1 mm above gel.
(5). Add 5.5 μl of 10X Antarctic Phosphatas buffer and 1 μl Antarctic Phosphatase to vector
digest. Mix by pipetting up and down, then incubate at 37 °C for 1 h.
(6.) Heat denature Antarctic Phosphatase by incubating at 65 °C for 10 min.
(7). Add agarose gel loading buffer to restriction digest. Load in more than two lanes. Run
gel at 75 V for 1 hr.
(8). Cut out vector DNA band (~5 kb) from gel while keeping cut piece to a minimal size
12
and extract DNA using the QIAquick Gel Extraction Kit (cat. no 28704). After adding 750
μl buffer PE, wait 3 min before proceeding to reduce salt content. Elute DNA in 30 μl buffer
EB. Use pipette tips with aerosol filter to avoid DNA cross-contamination. Wait for 2-3 min
after buffer EB addition to improve yield.
(9). Label tube with vector name “for ligation” and restriction enzymes used. Store at –20 or
–80 °C
2.1.2 Prepare insert DNA using PCR
(1). Dissolve primers: Spin down primer tube (1 min, top speed). Add MilliQ-H2O using
pipette tips w/ filter to give a primer concentration of 10 μM (e.g. 457 μl for 4.57 nmol). Let
stand for 1 min, then vortex for 30 s. Keep primers on ice and store at –20 or –80 °C.
(2). In PCR tube, add 22 μl of MilliQ-H2O, 1 μl of template DNA (e.g. vector DNA from
miniprep), 1 μl of forward primer, 1 μl of backward primer and 25 μl of 2X PfuUltra master
mix. Mix by pipetting up and down, use only tips with aerosol filter and keep everything on
ice.
(3). Run PCR program: 2 min at 98 °C, 35 cycles of 30s at 98 °C, 30s at annealing
temperature (e.g. 62 °C), 30s at 72 °C, followed by 10 min at 72 °C, then program to go to
20 °C infinitely.
(4). If PCR fragment is larger than 200 bp, extract DNA from PCR reaction using the
QIAquick Gel Extraction Kit. Elute DNA using 45 μl buffer EB. If PCR fragment is smaller
than 200 bp, skip this step.
(5). Transfer 42 μl of DNA solution into new tube, add 5 μl 10X restriction enzyme buffer,
1.5 μl restriction enzyme I, 1.5 μl restriction enzyme II, 0.5 μl 100X BSA. Mix by pipetting
up and down, then incubate at 37 °C for 3 h.
13
(6). If PCR fragment is larger than 200 bp, add agarose gel loading buffer and run
everything, depending on DNA size, on 30 ml of 1.5 to 2% agarose gel (heat and stir well!,
add ethidium bromide to 0.5 μg/ml to cooled agarose after boiling before pouring; use TBE
buffer; fill to 1 mm above gel and run at 75 V). Run in no more than three lanes.
(7). Cut out bands of correct size for all used lanes and extract DNA using the QIAquick
Gel Extraction Kit. Try to keep cut out piece to a minimum, i.e., do not cut out more than
necessary. Elute DNA in 30 μl of buffer EB. Use pipette tips with filter to avoid DNA cross-
contamination. Wait 2-3 min after buffer EB addition to improve yield.
(8). Keep insert on ice, label and store at –20 or –80 °C.
2.1.3 Ligate insert into cut vector
(1). Prepare a new tube, add 3 μl of prepared insert DNA and 3 μl of prepared vector. Then
to each tube add 11 μl MilliQ-H2O, 2 μl 10X T4 DNA Ligase reaction buffer and, finally, 1
μl T4 DNA Ligase. Mix by pipetting up and down using a 15 μl pipette setting, then
incubate at 16 °C o/n.
(2). Chill ligations mix on ice, then use 5 μl of ligation mix to transform XL-10 cells using
the lab’s transformation protocol. Use SOC medium and preheated (37 °C) LB Lennox agar
plates.
(3). Pick two single colonies from the plate with the most colonies, grow each one in 4.5 ml
LB medium in 14 ml Round Bottom Tube w/ Snap Cap tube containing the appropriate
antibiotic(s) o/n at 37 °C, 225 rpm.
(4). Load 2 ml of o/n culture in 2 ml tube and spin down (2 min, 2500g). Repeat once more,
then extract plasmid DNA from cell pellet using the QIAGEN Miniprep kit. Elute DNA
using 50 μl buffer EB. Remember to use new pipette tip for each clone.
14
(5). Transfer 4 μl (600 ng) into an appropriately labeled 0.5 ml PCR tube. Add 13.3 μl
MilliQ-H2O and 0.7 μl (6.4 pmol) of sequencing primer (10 μM stock). Use ‘colidown’
primer in PET44 vector, use AOX1 primer in ppic9k and ppiczα vector.
(6). Store all correctly sequenced clones at –80 °C and throw away wrong ones.
(7). Prepare backup, working vector DNA by growing a single colony of the desired clone
from the plate prepared in step 5 in 25 ml of LB medium containing the appropriate
antibiotic(s) for no longer than 16 h at 37 °C. Extract plasmid DNA by doing two minipreps
using 4 ml of cells for each one. Pool the two minipreps to give ~100 μl of new vector
DNA. Keep DNA on ice and store at –20 or –80 °C.
2.2 Overexpression of fusion protein-PET44-His6-TEV-xxx constructs
(1). Transform BL21(DE3) pLysS, T1R with pET44-GB3R2-xxx and plate on 100 μg/mL
ampicillin, 50 μg/mL chloramphenicol plate. Do not incubate plate for longer than 16 h at
37 °C. Use plate for max 7 days.
(2). First thing in the morning, inoculate a 2 ml LB Lennox culture containing 100 μg/mL
Amp, 34 μg/mL Chlor with a single colony and grow until dense at 37 °C and 200 rpm
(approx. 6-8 h).
(3). Then, per 1 L of expression culture, inoculate 25 ml starter minimal medium culture
containing 50 μg/mL Amp, 34 μg/mL Chlor with 0.25 ml of LB culture (1%) and grow
overnight at 37 °C and 200 rpm. Do not use more than 25% of flask volume. An expression
scale of 3 L is typical.
(4). On next morning, spin down the overnight minimal medium (MM) culture (5 min, 2500
g, room temp). Balance tubes to ±0.2g. Drain liquid completely and resuspend cells in 10
ml of new minimal medium by pipetting up and down.
15
(5). Inoculate minimal medium expression culture containing 50 μg/mL Amp, 34 μg/mL
Chlor with resuspended o/n MM culture and grow at 37 °C, 180 rpm. Use resuspended cells
worth of 25 ml of overnight culture per 1 L of fresh culture (2.5%).
(6). At an OD 600 of 1.0 induce protein expression with IPTG at a final concentration of 0.5
mM and grow for 4 h at 37 °C. Program shaker to go to 4 °C, 40 rpm afterwards.
(7). On next morning, harvest the cells by centrifugation at 4 °C and 4000g for 20 min.
Balance tubes to ±0.2g. Best, sometimes mandatory, not to freeze the cells.
(8). Resuspend cells in 30 ml per L culture volume of lysis buffer: 50 mM Na2HPO4
/NaH2PO4 pH 8.0, 300 mM NaCl (17.54 g/L), 20 mM imidazole (1.36 g/L), 100 mM SDS.
Stir with 10 ml pipette, cell clumps are ok, but keep cells on ice at all times.
(9). Add beta-mercaptoethanol to the cell suspension for a final concentration of 2 mM.
That is 140 μl/L from the 14.3 M beta-mercaptoethanol bottle in the hood.
(10). Sonicate on ice in <50 ml volumes in a 100 ml plastic beaker at level 7 until no longer
viscous. Sonicate in 45 s on, 60s off intervals for a total of 3-4 min of on time. Solution
should get warm, but not hot.
(11). Charge 5 ml HiTrap IMAC HP column with 5 ml of 0.1 M NiSO4 at 2 ml/min. Wash
out unbound Ni 2+ with 5 CV of MilliQ-H2O (This step is critical as NaP will precipitate
unbound Ni 2+). Then equilibrate column with wash buffer I, 50 mM Na2HPO4 /NaH2PO4
pH 8.0, 300 mM NaCl, 25 mM SDS.
(12). Centrifuge for 20 min at 20 °C at 20,000 rpm (SS-34 rotor). Balance tubes to ±0.1g.
Filter through coarse filter paper (VWR cat. no. 28331-026; 25 µm) in funnel on 100 ml
cylinder.
(13). Load supernatant on 5 ml HiTrap IMAC HP column at 1.0 ml/min. Collect and keep
16
flow-through.
(14). Wash with approx. 30 column volumes (CV) of wash buffer I beyond the point at
which a stable baseline is reached. When E 280 starts to drop, change flow rate from 1 to 3,
then to 5 ml/min.
(15). Wash out SDS for at least 15 CV (75 ml) with wash buffer II: 50 mM Na2HPO4/NaH
2 PO 4 pH 8.0, 300 mM NaCl (17.54 g/L), 8 M urea (480 g/L), 20 mM imidazole (1.36 g/L;
no need to readjust pH to 8.0).
(16). Wash off unspecific Ni 2+ -binder with wash buffer III, 50 mM Na2HPO4 /NaH2PO4
pH 8.0, 300 mM NaCl, 8 M urea, 50 mM imidazole, until a stable baseline is reached.
Collect this fraction.
(17). Elute GB3R-xxx in one tube (7-10 ml) using 50 Na2HPO4 /NaH2PO4 pH 8.0, 300
mM NaCl, 8 M urea, 300 mM imidazole (readjust pH to 7.4 after imidazole addition).
(18). Wash column with water and, for long term storage, equilibrate in 20% ethanol and
store at 4 °C. Do “System Wash Method” on ÄKTA with MilliQ-H2O.
(19). Dialyze eluted protein overnight against 5 L of 50 mM Tris pH 8.0, 0.5 mM EDTA pH
8.0, 0.5 M urea using a Spectra/Por 3 Dialysis Membrane, MWCO 3500, 45 mm (cat. no.
S632724). If a lot of protein has eluted, it is best to dilute the eluate 2-3 times with 50 mM
Tris pH 8.0, 8 M urea before dialysis.
(20). On next morning, take out dialyzed solution and rinse membrane with 2-3 ml of
dialysis buffer to get out all protein. Measure MBP/GB3R2-xxx concentration (e
280nm
= xxx
M
-1
cm
-1
; MW= yyy kDa). Blank against dialysis buffer.
(21). Add DTT to a final concentration of 1 mM and digest with TEV protease at a molar
ratio of 1:200 at 30°C overnight. Most cleaved peptides, but not all, are hydrophobic and
17
will appear as a white precipitate.
(21). On the next morning, collected precipitate by centrifugation (10 min, 4600 rpm). Run
a gel of supernatant. If there is still uncleaved GB3R2, re-measure protein concentration of
supernatant, add DTT and TEV again to cleave remaining GB3R2-xxx. If all protein is
cleaved, proceed to step 23 for supernatant and to step 24 for pellet. Most protein will be in
pellet.
(22). Wash precipitate with MilliQ water. Take up precipitate in, depending on size, 2-10 ml
of 50 mM Na2HPO4/NaH2PO4 pH 8.0, 300 mM NaCl (17.54 g/L),100 mM SDS. Can heat
sample to 40-50°C to dissolve completely.
(23). If not green any more, charge 5 ml HiTrap IMAC HP column (cat no. 17-0409-03)
with 5 ml of 0.1 M NiSO4 at 2 ml/min. Then equilibrate HiTrap IMAC HP column with 50
mM Na2HPO4/NaH2PO4 pH 8.0, 300 ml NaCl, 25 mM SDS at 5 ml/min.
(24). Load 3 ml of resuspended sample or supernatant in 5 ml superloop and inject at 0.2
ml/min on column. Collect peptide as flow through. For large volumes such as supernatant,
load through pump (take-off weight/filter on tube; consider the 12-13 ml of tube volume
before column is reached). Be careful not to suck in air into solvent line.
(25). Measure peptide concentration (e
280nm
= xxx M
-1
cm
-1
; MW= yyy kDa). Blank against 50
mM Na2HPO4/NaH2PO4 pH 8.0, 300 mM NaCl (17.54 g/L),100 mM SDS. Calculate
amount of peptide obtained per 1 L of expression culture.
(26). Elute bound proteins (fusion protein and TEV) with 50 mM Na2HPO4/NaH2PO4 pH
8.0, 300 ml NaCl, 8 M urea, 300 mM imidazole. Wash column with MilliQ-H2O and store
in 20% ethanol.
(28). Set Hamilton PRP-3 (305X7.0mm, cat. no. 79468) or Zorbax C18 column temperature
18
to room temperature or to 60 °C and turn on degasser.
(29). Prime pumps with A, 0.1% TFA in MilliQ-H2O and B, 0.1% TFA, 80% acetonitrile,
20% n-propanol (1-propanol) using the program “prime_pumps_AA” or
“prime_pumps_AB”. If new solvent bottle has been used and air entered the solvent line,
manual purging has to be performed.
(30). Set injection valve to “inject” and equilibrate column and sample loop with buffer A.
Use programs “Load_AA” or “Load_AB”.
(31). Filter sample through a Minisart SRP 15 0.20 μm filter. Set injection valve to “load”
and load the sample. Load no more than 5 ml into the 10 ml superloop. To inject, set
injection valve to “inject”.
(32). Start desired gradient elution program. For example, gradient from 30 to 70% B in 30
min at 3 ml/min (program “30_70_30min_3ml_AA” or “30_70_30min_3ml_AB”). Adjust
gradient start/stop and slope to give a symmetrical and well separated peptide peak.
(33). Collect peptide peak (can collect 1st and 2nd half separately).
(34). Turn off column heater. Either run program “column_wash” (from 100% MilliQ-H2O
to 50% acetonitrile, 50% n-propanol in 30 min and back, followed by pump water storage)
or “store_pumps” for storage in water.
(35). Freeze in liquid N2 for 10-15 min and freeze-dry. Do not fill tube more than half
(otherwise bottom section may thaw during freeze-drying).
2.3 Bicelles mimics test
(1) αIIb(A963C) and β3(G690C)-containing proteins were mixed in a volume of 100 μl at final
concentrations of 10 μM each in 50 mM Tris·HCl, pH 8.0 and 0.5 mM EDTA.
(2) The solution contained the bicelles depicted at a molar ratio of αIIb:β3:short-chain
lipid/detergent:long-chain lipid of 1:1:2000:600.
19
(3) Spontaneously formed cystines were reduced by adding tributylphosphine (TBP) or Tris(2-
carboxyethyl) phosphine (TCEP) to final concentrations of 5 mM and incubating for 2 hr at 30 °C
(Figure S1A).
(4) Disulfide bond formation was accelerated by adding the hydrophobic oxidant
Cu2+·(phenanthroline)2 to 2.5 mM and was allowed to proceed at room temperature for 1 hours
(5) The Cu2+·(phenanthroline)2 complex was prepared freshly by mixing equal volumes of 200 mM
CuSO4 in water and 400 mM 1,10-phenanthroline in N,Ndimethylformamide.
(6) To disintegrate the Cu2+·(phenanthroline)2 complex and block any remaining free cysteines, EDTA
and N-ethylmaleimide (NEM) were added to final concentrations of 10 and 2 mM, respectively.
(7) Subsequently, SDS-PAGE was performed using 4-20% Tris-Hepes or 4-20% Tris-Tricine
gels (Nusep, Inc.).
2.4 Transformation of Construct DNA into Pichia Pastoris

2.4.1 Linearizing Pichia DNA before transformation
(1) Use PME1 enzyme to linearize (from New England Biolabs Cat# R0560L)
(2) Mix the following in an Eppendorf tube. 10 µg circular DNA (eg: 100 µL at 200 µg/mL),
10 µL PME1 enzyme, 5 µL PME1 buffer (CutSmart buffer), 65 µL total volume.
(3) Adjust volumes accordingly depending on concentration of DNA
(4) Mix everything on ice.
(5) Incubate tube at 37
o
C for 1 hour in a water bath or heat block.
(6) Next purify the linearized DNA by precipitation with sodium acetate & EtOH.
(7) Add 1/10 (6.5 µL) volume of cold 3M NaAC, pH 5.2 to tube with linearized DNA
products, mix and give it a quick low speed spin.
(8) Add approx. 3X volume (195 µL) of 100% EtOH cold. Mix and incubate on ice for 10
minutes.
20
(9) Spin down at 11,000 rcf for 10 minutes, cold. Discard supernatant.
(10) Resuspend in 500 µL 70% EtOH (room temp is fine), Spin down again at 11,000 rcf for
10 minutes, cold.
(11) Vacuum off solution until edges of pellets appear a little translucent. Pellets should be
just a little wet.
(12) Add 35 µL H2O to pellets. Gently tap to resuspend. Note: adjust amount of H2O added
to reach desired concentration for transformation (depends on amount of DNA present).
(13) Measure concentration of DNA and keep cold until ready to transform.

2.4.2 Electroporation of competent cells with linearized DNA
(1) Make sure DNA is linearized and purified.
(2) Take out 50 µL of competent cells (bg12 His 4-) from the – 80
o
C fridge and thaw it on
ice.
(3) Add 35 µL of linearized DNA. Then, mix by pipetting up and down several times.
(4) Pipette the (competent cells/ linearized DNA) mixture into the bottom of the cuvette.
(5) Put cap on cuvette and incubate on ice for 10 minutes.
(6) Set Bio Rad micropulser. 1.5kv 200ohms 25 uF 2mm
(7) Zap and then immediately add 1 mL cold 1 M sorbitol.
(8) Pipette the cell/sorbitol slurry into culture tube and incubate at 30
o
C for 1 hour and 200
rpm.
(9) Spin down culture at 5000 g for 5 minutes, remove 750 µL supernatant.
(10 ) Pipet up and down to resuspend cells with the remaining supernatant.
(11 ) Pipet cells onto prewarmed YNB agar plate.
21
(12) Incubate plate at 30
o
C for 2-3 days.
2.5 Expression test of construct
(1) At 3:00pm, using a single colony, inoculate 4 mL of BMGY in 13 mL culture tube. Grow
at 30 o C in a shaking incubator at 200 rpm overnight to reach OD=2-6 (approx.16-18
hrs).
(2) The next morning at 8:30am, measure OD of culture and spin it down at 2,500xg for 5
mins.
(3) Discard supernatant. Resuspend the cell pellet to an OD=1.0 using 4 mL of BMMY to
induce protein expression. Grow the culture for 24 hours.
(4) The next morning at 8:30am, add 100% methanol to obtain a final concentration of 0.5%
to maintain induction. Grow the culture for 24 hours.
(5) The next morning at 8:30am, take 1 mL culture and spin it down at 15,000 rpm in an
Eppendorf tube. Save supernatant. Run Western Blot.
2.6 Western Blot
2.6.1 SDS-PAGE
(1) Run SDS-PAGE with the normal procedure.
(2) Once finished running the gel, cut off the gel on the top that used to form the well.
2.6.2 Transfer SDS-PAGE to Nitrocellulose membrane
(1) Prepare 1L of 1x NuPage transfer buffer with 20% methanol.
(2) Soak the two black sponges and 10 pieces of filter paper, cut to be a fraction smaller than
the sponges, in the transfer buffer at 4
o
C.
(3) Using the BioRad equipment (green top, red and black case), assemble the “sandwich”
holder by placing one of the soaked sponges onto the black side of the holder.
22
(4) Add on 5 pieces of the soaked filter paper and using a 10 mL pipette, roll over the paper
to get rid of any air bubbles.
(5) Next put the gel on top of the filter paper.
(6) Lay the nitrocellulose membrane on top of the gel. Make sure don’t move the membrane
once on top of the gel.
(7) Add 5 more pieces of the soaked filtered paper, and once again roll out the air bubbles.
(8) Place the second wet sponge on top of the filter paper and close the sandwich holder.
(9) Place the holder into the plastic container with the red and black apparatus already inside.
The gel should be closest to the black, while the nitrocellulose membrane should be
closest to the red side.
(10 ) Put the ice block in the white container into the western blot apparatus container and
fill it with approx. 1000 mL of transfer buffer.
(11 ) Put on the lid and run at 100V for 60 minutes.
(12 ) Afterward, you should see that the Pre-stained marker ladder transferred from the SDS-
PAGE to the membrane.
2.6.3 Blocking
(1) Mark the gel at the corner so you know it’s correct orientation.
(2) Soak the membrane in 5% (by weight) dry milk in TBST buffer. Agitate it for 1 hour at
room temperature or overnight at 4
o
C with slow agitation. (To make 10X TBST stock
solution, dissolve 80g NaCl, 24g Tris, 100 µL tween 20 in 800 mL MQ water, then adjust
the pH to 7.6 using HCl under the monitoring of the pH meter, bring the volume up to
1000mL).
2.6.4 Immunostaining
23
(1) Pour out the blocking solution (milk) and wash the membrane with 30 mL 1X TBST for 5
minutes with agitation. Repeat 2 more times.
(2) Pour out the TBST buffer and add 10 mL of the first antibody solution. Agitate at room
temperature for 1 hour or overnight at 4
o
C. (To make 10 mL of antibody solution, add 7
mL of 1X TBST, 0.3g BSA, 10 µL Monoclonal ANTI-FLAG® M2-Alkaline Phosphatase
antibody, mix to dissolve, then bring the volume to 10 mL, and mix thoroughly).
(3) Pour off the first antibody into a 15 mL tube and save it for future use (It can be reused
for up to 3 times.) Wash the membrane with 30 mL 1X TBST for 5 minutes with
agitation. Repeat 2 more times.
(4) Pipette 3 mL CDP-Star substrate into a 15 mL tube, then add 150 µL Nitro-Block-II
enhancer, vortex briefly.
(5) Put the nitrocellulose membrane in a small container (protein side up), then pipette the
solution in step 4 onto the surface of the nitrocellulose membrane. Incubate at room
temperature for 5 minutes.
(6) Take the nitrocellulose membrane to 2
nd
floor room 234. Locate the Syngene G:BOX
imaging machine. Double click GenSys icon on the desktop computer. Wait 5 seconds for
the program to turn on. Click “Blot,” then choose Chemi Blot (single Image). On Dye
selection, choose CDP-Star. Note: a message will pop-up. “Please insert the Black Anti-
reflective Screen into the darkroom then press OK.”
(7) Put nitrocellulose membrane in the center of the Black Anti-reflective Screen, then insert
it into the imaging machine. Press OK. Press Next, then press Capture. Save image.

24
CHAPTER 3: RESULTS
3.1 Interaction of the cytosolic and transmembrane domain between αIIb and β3
NMR spectroscopy is highly suitable to contribute to the structural and dynamic
characterization of transmembrane and cytosolic complex of αIIbβ3 when a membrane mimic that
supports interactions can be identified.

Figure 7: Illustration of fusion proteins used for αIIb (Ala963Cys) and β3 (Gly690Cys)
transmembrane and cytosolic peptides.
A high-throughput approach to quantify the function of membrane mimics parameter has
been developed before. The heterodimeric integrin αIIbβ3 cytosolic and transmembrane complex
is used as model system for the association of transmembrane and cytosolic peptides. The
physiological interactions can lead to αIIb-β3 association. Then, it is hypothesized that the relative
suitability of a membrane mimic can be judged by the amount of heterodimer obtainable in its
presence.
25
To detect αβ dimer quantities, SDS−PAGE was employed. To ensure the αβ dimer
association and the stabilization of the complex in following NMR studies, αIIb (Ala963Cys) and
β3 (Gly690Cys) mutant construct were introduced to allow the covalently cross-link of the
heterodimer. To distinguish between the αIIb-αIIb and β3-β3 homodimers in the experiment, we
introduced a large molecular weight fusion protein MBP into the αIIb. In vitro, the αIIbβ3
transmembrane complex had been studied in organic solvent, 20 detergent micelles,21 and
isotropic bicelles. In our study, we lay great importance on comparing between the different
combinations of bicelles. 6 types of bicelles were evaluated.

Figure 8: SDS-PAGE results of different bicelles combination.
1.CHAPSO/DMPC/DMPG 2. CHAPS/DMPC 3. CHAPS/DMPG 4. DHPC/POPC 5.
CHAPSO/DMPC 6. CHAPS/POPC

26
a.

b.

Figure 9. Relative quantities of accumulated αIIbβ3 cytosolic and transmembrane species
as a function of membrane mimic. a. Dimer obtained with MBP-αIIb (Ala963Cys) and
β3(Gly690Cys) proteins. The size of each color-coded bar denotes its molar ratio among α, αα,
ββ, and αβ species. For visual clarity, explicit bars were omitted for α and β. b. Species obtained
with MBP-αIIb (Ala963Cys) and β3(Gly690Cys) proteins. The size of each color-coded bar
approximates the mass distribution of MBP-αIIb among α, αα, and αβ species. In all
27
experiments, the molar ratio of αIIb: β3: short-chain lipid/detergent:long-chain lipid was
1:1:2000:600 with protein concentrations of 10 μM.
From the above results, we concluded that CHAPSO/DMPC has the largest αIIbβ3 ratio
and DHPC/POPC, CHAPS/POPC has the highest band intensity. In the following NMR
experiment sample preparation, either one of them can be used.
To test the interaction between the αIIbβ3 cytosolic and transmembrane domain, NMR
titration groups have been set as in table 1. With the different combination between αIIb
transmembrane and cytosolic domain and β3 transmembrane and cytosolic domain, the interaction
dynamics could be studied.

Table 1: NMR titration group for αIIbβ3 cytosolic and transmembrane domain.
28

Figure 10: Chemical Exchange Mode in NMR. k: reaction rates. Δf: chemical shift
time scale

In NMR process, chemical exchange is relevant both in the situation where a protein
undergoes internal motions such as a conformational change) or where it interacts with another
molecule (complex formation). Each conformation will have a distinct isotropic value of chemical
shift. If the rate at which the conformation switches is slow compared to the duration of the NMR
experiment (typically in the millisecond regime) then two separate peaks will be observed in the
NMR spectrum. However, as the exchange rate increases, the molecule will start to switch between
conformations one and two, then each become modulated by the other. The end effect is that each
peak in the spectrum to move and become broader because the nuclei in the sample no longer all
have chemical shift of one state(21). Previous NMR spectroscopy of only transmembrane domain
shows the peaks corresponding to the αIIb monomer and αIIbβ3 dimer are in slow exchange.
29
Therefore, in principle, distinct peaks corresponding to the monomer and dimer can be
observed. However, in our NMR spectroscopy of transmembrane and cytosolic domain, only
single signal is observed with intermediate chemical shift. In this situation, the following
experiments have to be terminated.

3.2 αIIbβ3 extracellular domain expression
3.2.1 Construct design

a b c
Figure 11: Crystal structure of the chosen extracellular domain (green). a. αIIb (L1-
P452) b. β3(V58-D434) c. β3(D109-K354)
The major problem of the expression of αIIbβ3 extracellular domain is their large
molecular weight, which is not accessible with normal E. coli system. Vectors pPICZα and
pPic9k from Pichia Pastoris were adopted. pPICZα-alpha-mating-αIIb (L1-P452)-FLAG-His6
and pPIC9K-alpha-mating-β3(V58-D434)/β3(D109-K354)-FLAG-His6 are the first construct
groups. At the meantime, construct pPic9k-alpha-mating-KREAEA-FLAG-CD33(D140-T232)
C169S-His6-TEV has been proved to be expressed and secreted well by my coworkers.
Therefore, it is inferred that inserting my target protein construct into this well-expressed
construct should help my target protein to secret. Three constructs are designed as pPic9k-
alpha-mating-KREAEA-FLAG-CD33(D140-T232) C169S-His6-TEV-αIIb(L1-P452) and
30
pPic9k-alpha-mating-KREAEA-FLAG-CD33(D140-T232) C169S-His6-TEV-β3(V58-
D434)/ β3(D109-K354).

Figure 12: Plasmid Map of pPICZα.

Figure 13: Plasmid Map of pPIC9k.
3.2.2 Plasmid construct process of pPICZα-KR-alpha-mating-αIIb (L1-P452)-FLAG-His6 and
pPic9k-alpha-mating-KREAEA-FLAG-CD33(D140-T232) C169S-His6-TEV- αIIb (L1-
P452).
31
(1) Make silent mutation from CTC GAG to CTC GAA of pEF1-αIIb to remove XhoI
restriction enzyme site.
(2) Make silent mutation from AGC GGC to AGC GGA of step 1 to remove NotI restriction
enzyme site.
(3) Clone out αIIb (L1-P452) (Template: pEF1-αllb XhoI Removed), cut with XhoI/EcoRI to
isolate insert for pPICZα-alpha-mating-αIIb (L1-P452)-FLAG-His6; cut with EcoRI/NotI
to isolate insert for pPic9k-alpha-mating-KREAEA-FLAG-CD33(D140-T232) C169S-
His6-TEV- αIIb (L1-P452).
(4) Cut pET44-alpha-mating with XhoI/ EcoRI to isolate vector.
(5) Do ligation to obtain pET44-alpha-mating-αIIb (L1-P452).
(6) Cut pET44-alpha-mating-αIIb(L1-P452) with BamH1/EcoRI to isolate insert.
(7) Cut pPICZα with BamH1/EcoRI to isolate vector; cut pPic9k-aM-KREAEA-FLAG-
CD33(D140-T232) C169S-His6-TEV with EcoRI/NotI to isolate vector.
(8) Do ligation to obtain pPICZα-alpha-mating-KR-αIIb (L1-P452)-FLAG-His6; do ligation
to obtain pPic9k-alpha-mating-KREAEA-FLAG-CD33(D140-T232) C169S-His6-TEV-
αIIb (L1-P452).
αIIb (L1-P452) XhoI
removed
αIIb (L1-P452)

αIIb (L1-P452) XhoI
and NotI removed
Forward
Primers
5'-TTGCGGATATT
TTCTCGAGTTAC
CG CCCAGGCATC-
3'
5'- GATATACTCGAG
AAGAGAGATTATAA
GGATGACGACGATA
AATCTGGTTTGAACC
TGGACCCAGTG-3'
5'-CCAGAGAGCGG
CCGCCGCGCCGA-3'
Backward
Primers
5'-GATGCCTGGGC
GGTAATTCGAGAA
AATATCCGCAA -3'

5'- AAGGGCGAATTCT
TAATGGTGATGATGG
TGATGTGAACCTGGC
TGAGCTCTGTACACAG
-3'
5'-TCGGCGCGGCGT
CCGCTCTCTGG-3'
Table 2: Primer design of αIIb (L1-P452).
32
3.2.3 Plasmid construct process of pPIC9K-alpha-mating-β3(V58-D434) / β3(D109-K354)-
FLAG-His6 and pPic9k-alpha-mating -KREAEA-FLAG-CD33 (D140-T232) C169S-
His6-TEV- β3(V58-D434)/ β3(D109-K354).

(1) Clone out β3(V58-D434)/ β3(D109-K354). from template: pcDNA3.1- β3), cut with
XhoI/EcoRI to isolate insert.
(2) Cut any pET44-alpha-mating-KR-CD33 construct with XhoI/ EcoRI to isolate vector.
(3) Do ligation to obtain pET44-alpha-mating-KR- β3(V58-D434)/ β3(D109-K354).
(4) Cut pET44-alpha-mating-KR- β3(V58-D434)/ β3(D109-K354). with BamH1/EcoR1 to
isolate insert.
(5) Cut pPic9K with BamH1/EcoR1 to isolate vector; cut pPic9k-alpha-mating-KREAEA-
FLAG-CD33(D140-T232) C169S-His6-TEV with EcoRI/NotI to isolate vector.
(6) Do ligation to obtain pPic9K-alpha-mating-KR- β3(V58-D434)/ β3(D109-K354).; do
ligation to obtain pPic9k-alpha-mating-KREAEA-FLAG-CD33(D140-T232) C169S-
His6-TEV- β3(V58-D434)/ β3(D109-K354).

β3(V58-D434) β3(D109-K354)
Forward
Primers
5'-GATATACTCGAGAA
AAGAGATTATAAGGA
TGACGACGATAAATC
TGGTGTGAGTGAGGC
CCGAGTACT-3'
5'-GATATACTCGAGAAA
AGAGATTATAAGGATGA
CGACGATAAATCTGGTGA
TTACCCTGTGGACATCTA
CTAC-3'
Backward
Primers
5'-AAGGGCGAATTCTTA
ATGGTGATGATGGTGAT
GTGAACCTCACAATCAA
AGGTGACCTG -3'
5'-AAGGGCGAATTCTTAA
TGGTGATGATGGTGATG
TGAACCTTTAGAACGGA
TTTTCCCATA-3'
Table 3: Primer design of β3(V58-D434) / β3(D109-K354).

33
3.2.4 Western Blot Results

a.

b.

34
c.
Figure 14: Western blot results. a. pPic9k-alpha-mating-KREAEA-FLAG-CD33(D140-
T232) C169S-His6-TEV-αIIb (L1-P452) b. pPic9k-alpha-mating -KREAEA-FLAG-
CD33(D140-T232) C169S-His6-TEV- β3(D109-K354). c. pPic9k-alpha-mating -KREAEA-
FLAG-CD33(D140-T232) C169S-His6-TEV-β(V58-D434).

However, above results show that no proteins were expressed of pPic9k-alpha-mating-
KREAEA-FLAG-CD33(D140-T232) C169S-His6-TEV-αIIb (L1-P452) and pPic9k-alpha-
mating-KREAEA-FLAG-CD33(D140-T232) C169S-His6-TEV- β3(D109-K354) constructs.
(Figure A and B). Two lanes of construct pPic9k-alpha-mating-KREAEA-FLAG-
CD33(D140-T232) C169S-His6-TEV- β3(V58-D434) are not our target protein due to the
unmatched molecular weight.

35
a.
b.
c.
Figure 15: Western blot results. a. pPIC9K-alpha-mating-β3(D109-K354)-FLAG-His6 b.
pPIC9K-alpha-mating-β3(V58-D434). c. pPIC9K-alpha-mating-β3(V58-D434) with reducing
agent.
36
Unfortunately, above two results also show that no protein was expressed in construct
pPIC9K-alpha-mating-β3(D109-K354)-FLAG-His6 and pPIC9K-alpha-mating-β3(V58-D434).
Then the screening of construct pPICZα-alpha-mating-αIIb (L1-P452)-FLAG-His6 was cancelled
due to the non-ideal results of β3 construct.
3.3 αIIb and αV Thigh-calf1-calf2 domain expression
3.3.1 Construct design
a. b.
Figure 16: Crystal structure of designed construct. a. αIIb red-thigh/yellow-calf1/blue
calf2 b. αV blue-thigh/yellow-calf1/grey-calf2.

αIIb Thigh-Calf1-
Calf2
αIIb Thigh-Calf1

αIIb Calf2
Forward
Primers
5'-GATATAGCTC
GAGATGCTCAGCC
GGTTGTTA
AAGC-3'
5'-GATATAGCTCG
AGATGCTCAGCC
GGTTGTTAAAG-3'

5'-GATATAGCTCGAG
ATGAAGCGCAGGTT
GAACTG 3'

Backward
Primers
5'-AAGGGCGGAA
TTCCCACGTTCTTC
CAGCGCAC-3'
5'-AAGGGCGGA
ATTCCCTTCCGCACG
AACCGG
-3'
5'-AAGGGCGGAAT
TCCCACGTTCTTCCA
GCGCAC-3'
Table 4: Primer design of αIIb leg domain.

37
αV Thigh-Calf1-Calf2 αV Thigh-Calf1

αV Calf2
Forward
Primers
5'-GATATAGCTCGA
GATAGACCAGTTAT
CACTGTAAATGCT-
3'

5'-GATATAGCTCG
AGATAGACCAGTTA
TCACTGTAAATGCT
-3'

5'-GATATAGCTCGA
GATGTTTTAGCTGCG
TTGAGATAAG-3'

Backward
Primers
5'-AAGGGCGGAAT
TCCCGCCCCAGGTG
ACATTAGTG-3'
5'-AAGGGCGGAAT
TCCCAGCAAGATCA
ACTTTGTGAGATAC-
3'
5'-AAGGGCGGAAT
TCCCGCCCCAGGTGA
CATTAGTG-3'
Table 5: Primer design of αV leg domain.

The leg domain is composed of thigh, calf1 and calf2 different domains both in αIIb and
αV. We assumed that the protein folding process could be affected with the different combination
of three domains. Three different constructs were designed in each integrin: as pPic9K-Msb2-
XhoI-αIIb/αV Thigh-Calf1-Calf2-EcoRI-TEV-FLAG-His10, pPic9K-Msb2-XhoI-αIIb/αV
Thigh-Calf1-EcoRI-TEV-FLAG-His10 and pPic9K-Msb2-XhoI-αIIb/αV Calf2-EcoRI-TEV-
FLAG-His10. The failure of head domain expression leads us a new strategy of changing the
secreting signal sequence. Alpha-mating signal sequence is widely used in many protein
expression systems. However, it has been inferred from our former experiments results that the
alpha-mating signal sequence was not suitable for the expression of extracellular domain of
integrin. A new discovered signal sequence MSB2 (MINLNSFLILTVTLLSPA LA↓LPKNVLEE
QQAKDDLAKR) has been used instead based on the former success of expression with my
coworkers.

38
3.3.2 Western Blot Result

Figure 17: Western blot result. αIIb thigh-calf1-calf2
Unfortunately, there is no protein expressed in αIIb thigh-calf1-calf2 construct.
Following testing of other constructs will continue.

Chapter 4: Discussion
Integrin αIIbβ3 has important research value as a typical kind of heterodimer
transmembrane protein. While many different structures related to αIIbβ3 has been studied, their
interaction dynamics between transmembrane and cytosolic domain hasn’t been determined in
detail. In my study, we designed to conduct NMR titration experiments of the cytosolic and
transmembrane interaction between αIIb and β3. We have determined that CHAPSO/DMPC,
DHPC/POPC and CHAPS/POPC are the suitable bicelle mimics for NMR experiment. However,
the following titration experiment wasn’t successful due to the intermediate chemical exchange,
which provide us useful insight about improving the NMR testing limitations. To study the
unliganded extracellular domain structure dynamics in NMR, a series of expression events have
39
been conducted. We chose one construct of αIIb and two constructs of β3 with alpha-mating signal
sequence to express with pichia pastoris system, which has been proved to be available in various
proteins. However, all the construct we tested were failed. Among all the constructs, we
successfully tested two lanes of proteins in the pPic9k-alpha-mating-KREAEA-FLAG-
CD33(D140-T232) C169S-His6-TEV-b3(V58-D434), but with nearly half of the molecular
weight of our target protein. What has been possibly speculated is that some protease could be
functional during the secreting process. For our next step, protease inhibitors are a promising
solution. Protease inhibitors are often added to the lysis buffer and in early steps of the purification
process to prevent degradation of the target protein by endogenous proteases. For example, metal
chelating reagents, such as EDTA or EGTA, are often added to the storage buffer. These metal
chelators can bind to Mg2+ and prevent cleavage of the protein by contaminating metalloproteases.
Besides, it is also possible that our protein was successfully synthesized and expressed yet fail to
be secreted externally of the cell. For future study, lyse the cell interiorly and test the expression
again could be an accessible method. However, the failure to harvest the secreting proteins could
also be speculated as that the alpha-mating signaling sequence we used may be improper for the
secretion of our integrin αIIbβ3. Therefore, in the expression of the αIIb and αV leg domain, a
MSB2 signal sequence was inserted instead on account of its success in the expression of other
extracellular protein by my coworker. Yet the result of the one tested construct αIIb thigh-calf1-
calf2 was still disappointing. In conclusion, there are some proteins who are extremely hard to
express in heterologous expression systems. Different factors can address for this problem. It has
been said that a normal possibility is that a foreign host is not easily able to fold proteins correctly.
Moreover, internal properties of target protein can also be challenging for expression. Post-
translational modifications are one of the difficulties we may meet in our experiments. The real
40
modifications process is hard to be monitored even theoretically the pichia pastoris system
successfully allows the post-translational modifications. Currently there is no specific solution for
the expression of those troublesome proteins. Under this circumstance, my work of failed attempts
provided precious foundation for the future expression of integrin αIIbβ3 and all integrin
heterodimeric family proteins.

41
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Asset Metadata

Creator Xiao, Jiaqi (author)

Core Title Study of integrin αIIbβ3 transmembrane and cytosolic domain interaction and the expression of extracellular domain by Pichia Pastoris

Contributor Electronically uploaded by the author (provenance)

School Keck School of Medicine

Degree Master of Science

Degree Program Medical Physiology

Degree Conferral Date 2021-08

Publication Date 08/02/2021

Defense Date 05/17/2021

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

Tag intergrin,NMR,oai:digitallibrary.usc.edu:usctheses,OAI-PMH Harvest,Pichia Pastrois,protein expression,protein structure,transmembrane protein

Format application/pdf (imt)

Language English

Advisor Lange, Ralf (committee member), Siemer, Ansgar (committee member), Ulmer, Tobias (committee member)

Creator Email jiaqixia@usc.edu,jiaqixiao10@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC15672377

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Abstract (if available)

Abstract Integrin is a family of heterodimeric proteins which has small cytosolic, transmembrane, and large extracellular domain and could act through two mechanisms: inside-out and outside-in pathways. Upon agonist stimulation, the transduction of inside-out signals leads integrin αIIbβ3 to switch from a low- to high-affinity state for ligands, which is associated with the structure change of cytosolic and transmembrane domain. NMR analysis is the best way to study the αIIbβ3 complex structure change in binding. Apart from inside-out signaling, ligand binding can reverse promote outside-in signaling and drive essential platelet function, which is related to the structure of αIIbβ3 extracellular domain. The crystal structure of αIIbβ3 extracellular domain has been determined, however, it has never been studied in solution formation. Due to its large size, the Pichia Pastoris yeast expression system is employed to synthesize the extracellular domain instead of the E. coli system used in the cytosolic and transmembrane domain. The construction of the integrin αIIbβ3 model will precisely and accurately explain the role of integrin in transmitting extracellular signals into recipient cells and exporting cellular regulations to the external environment in blood hemostasis and thrombosis. The reveal of the real structure of integrin αIIbβ3 could also boost the development of new drugs and provide a solid experimental foundation for further structure determination with different integrin family proteins.