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Towards DNA-directed assembly of pMHC multimers for detection of low-affinity T cells
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Towards DNA-directed assembly of pMHC multimers for detection of low-affinity T cells
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Towards DNA-directed assembly of pMHC multimers for detection of low-affinity T
cells
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BY
Ting Fu
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A Thesis Submitted in Partial Fulfillment of
the Requirements for the Degree of
MASTER OF SCIENCE IN
MOLECULAR PHARMACOLOGY AND TOXICOLOGY
AT
UNIVERSITY OF SOUTHERN CALIFORNIA
May 2016
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Table of Contents
Acknowledgements III
List of Tables IV
List of Figures V
Abbreviations VI
Abstract VII
1. Introduction 1
1.1. Antigen recognition by T cells 1
1.2. Isolation of antigen-specific T cells using pMHC tetramers 4
1.3. Alternatives to pMHC tetramers 7
1.4. DNA-directed assembly of proteins 9
2. Results and Discussion 12
2.1. Synthesis and characterization of Y-shaped DNA 12
2.2. Expression, in vivo refolding, and purification of streptavidin 15
1) Evaluation of commercial streptavidin 15
2) Determination of inclusion body quality 18
3) Refolding of streptavidin 19
4) Purification of refolded streptavidin 27
5) Verification of the biotin-binding capability of homemade streptavidin 30
2.3. Conjugation reaction 32
2.4. Future plan 35
3. Method 37
3.1. Synthesis and characterization of Y-shaped DNA 37
3.2. Expression, in vitro refolding and purification of streptavidin 38
3.3. Conjugation reaction 41
References 43
! III!
!
Acknowledgements
I would like to take this opportunity to thank all of those, who have helped me with this
thesis as well as my studies at School of Pharmacy at USC.
First of all, I would like to express my sincere gratitude to my advisor, Dr. Jianming Xie,
who provided me with the opportunity to join his lab. I appreciate his expertise and
valuable guidance on all the experiments I have conducted for this research project.
Without his persistent guidance, this thesis would not be possible.
I would like to thank my committee members, Dr. Curtis Okamoto and Dr. Bangyan Stiles.
I am extremely grateful for their helpful suggestions and assistance throughout the whole
thesis project.
In addition, I am thankful for all the members in this lab for their advice, support, and
encouragement on my experiments during my research work at USC. I would like to
particularly express my sincere appreciation to Rebecca Lim for proofreading this thesis.
! IV!
List of Tables
Table 1. Troubleshooting trials for refolding of streptavidin 23
! V!
List of Figures
Figure 1. TCR-pMHC recognition 2
Figure 2. A Y-shaped DNA and Y-shaped DNA scaffold based pMHC nonamer 13
Figure 3. Characterization of Y-shaped DNA 14
Figure 4. Evaluation of two commercial streptavidin products on a 4%-12% Bis-Tris gel.
17
Figure 5. Evaluation of the mutant streptavidin inclusion body quality on a 4%-12%
Bis-Tris gel. 19
Figure 6. Detection of the refolded mutant streptavidin on a 4%-12% Bis-Tris gel 21
Figure 7. Detection of the refolded wild type streptavidin on a 8%-12% Tris-Glycine
protein gel 25
Figure 8. Purification of refolded wild type streptavidin through the Superdex 200 10/300
GL column 28
Figure 9. Purification of refolded wild type streptavidin through the MonoQ column 29
Figure 10. Conjugation of biotinylated ovalbumin to streptavidin 31
Figure 11. Conjugation of biotinylated oligonucleotide to streptavidin 34
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! VI!
Abbreviations
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TCR, T Cell Receptor
MHC, Major Histocompatibility Complex
pMHC, peptide-Major Histocompatibility Complex
K
D
, Dissociation Constant
CpG, Cytosine-phosphate-Guanine
TLR, Toll-like Receptor
SPR, Surface Plasmon Resonance
IPTG, Isopropyl β-D-1-thiogalactopyranoside
E.coli, Escherichia coli
SAV, Streptavidin
SDS-PAGE, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
PBS, Phosphate Buffered Saline
EDTA, Ethylenediaminetetraacetic acid
Fab, Fragment Antigen-binding
pI, Isoelectric Point
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! VII!
Abstract
T cells, which play a central role in cellular immunity, express distinct T cell receptors
(TCR) to recognize antigenic peptides bound to major histocompatibility complex
molecules (MHCs). The ability to detect, quantify, isolate, and characterize often very rare
antigen-specific T cells from a population is very important to both fundamental and
clinical immunology. Monomeric peptide-MHC (pMHC) molecules cannot stably stain
cognate T cells because the TCR–pMHC interaction has a low affinity (K
D
=1-100 µM)
and a fast disassociation half-life (t
1/2
= 1-10 s). This problem has been significantly
addressed by the development of pMHC tetramers and other forms of multimers, which
are capable of binding to multiple TCRs simultaneously. However, it has been known that
some T cells, e.g., cancer or auto-reactive T cells, can express extremely low-affinity
TCRs that are beyond the detection limit of pMHC tetramers. In this thesis, we aim to
develop a novel type of multimeric pMHC reagent that has significantly enhanced
detection sensitivity. Specifically, we attempt to use a Y-shaped DNA scaffold to present
three streptavidin molecules, which in turn will be conjugated to multiple biotinylated
! VIII!
pMHC molecules. The DNA-directed assembly of pMHC complexes will have two main
characteristics: 1) it is capable of presenting nine pMHCs in one complex; and 2) the
shape and length of the DNA scaffold can be conveniently manipulated to achieve optimal
binding avidity for TCRs on the T cell surface. As of now, I have already established a
protocol to synthesize and purify a Y-shaped DNA with one biotin at each of the three
arms. I have also tested more than a dozen in vitro refolding conditions and finally found
an optimal protocol to produce and purify the correctly refolded streptavidin protein.
Currently, I am optimizing the conjugation between the Y-shaped DNA and the
fluorescently labeled streptavidin. Our hope is that this novel DNA-streptavidin complex
will enable the presentation of a variety of pMHC class I and II molecules, of both human
and mouse origin, for the successful detection of low-affinity T cells in cancer and
autoimmune diseases.
! 1!
1. Introduction
1.1. Antigen recognition by T cells
T cells are a type of white blood cells that play central roles in many aspects of the
immune system. They express T cell receptors (TCRs) to scan antigenic peptides bound
to major histocompatibility complex (MHC) molecules on the surface of antigen
presenting cells. Depending on the type of T cells, recognition of antigens leads to
distinct immune responses: 1) cytotoxic T cells kill pathogen-infected cells or tumor
cells directly; and 2) helper T cells produce cytokines to stimulate other immune cells,
such as B cells, macrophages, and cytotoxic T cells, to respond to infections. T cell
responses can also become harmful, e.g., T cells that accidently target self organs cause
autoimmune diseases. Therefore, understanding how T cells recognize antigens in
health and disease is very important to both fundamental and translational immunology.
The TCR is a heterodimeric protein resembling the fragment antigen-binding (Fab)
fragment of an antibody. Specifically, it consists of an α chain and a β chain, and each
chain is composed of a variable and a constant extracellular domains. The constant
! 2!
domain is proximal to the cell membrane, whereas the variable region is away from the
cell membrane and forms the pMHC-binding surface. The MHC molecule is a cell
surface protein that presents short peptide fragments to be recognized by TCRs shown
in Figure!1. There are two types of MHC molecules: class I and class II. These MHC
molecules have distinct subunits, but share a similar three-dimensional structure. They
both form a peptide-binding cleft, where the antigenic peptide is bound and recognized
by TCRs.
Figure 1. TCR-pMHC recognition
!
T cells are very specific, able to discern a single amino-acid difference between
two pMHC molecules. T cells are also extremely sensitive-even a single pMHC
! 3!
molecule can induce T cell activation, as shown by using high resolution fluorescence
microscopy as well as the quantum dot technique. The high sensitivity of T cell
recognition is likely due to the capability of a single pMHC to trigger 180 TCRs
serially (the serial triggering model). (VALITUTTI et al., 1995) The successive
activation of multiple TCRs by one pMHC could be attributed to the fast dissociation
rate and low affinity of the TCR-pMHC interaction. The surface plasmon resonance
(SPR) analysis has showed that the TCR recognition of antigenic pMHC has a K
D
of
1-100 µM, which is 1,000-10,000 times weaker than that of antibody recognition of
antigens.(Huppa et al., 2010) In addition, the TCR-pMHC interaction has a fast off-rate,
having a t
1/2
on the order of seconds.(Matsui et al., 1994) A recent analysis using
super-resolution fluorescence imaging suggested that TCRs are enriched in separate
membrane domains (protein islands) with a diameter of 30-300 nm on the surface of T
cells. Thus it is a likely scenario that a pMHC molecule recognized by a TCR can
quickly dissociate and move to activate the next TCR in the same protein island.
(Lillemeier, 2010)
! 4!
1.2. Isolation of antigen-specific T cells using pMHC tetramers
While each T cell only bears a single antigen specificity by expressing distinct
TCRs, the total TCR diversity is huge resulting from the somatic recombination process.
Previous TCR gene sequencing studies have suggested that the theoretical TCR
diversity can reach 10
18
,(Davis and Bjorkman, 1988) and the estimated 10
12
T cells in
the human body express about 2.5x10
7
distinct TCRs(Arstila et al., 1999). Upon
encountering pMHC ligands, a small number of cognate T cells will be activated and
undergo clonal expansion. Therefore, the ability to detect, quantify, isolate, and
characterize antigen-specific T cells is of critical importance to a broad range of
immunological studies.
Flow cytometry provides a direct and quantitative method to enumerate and isolate
the small fraction of cells stained with fluorescence-labeled specific antigens. The high
affinity of B cell receptors (BCRs) for their antigens has allowed wide applications of
this technique on the investigation of B cells, such as development of memory B cells
in vivo.(McHeyzer-Williams et al., 1991) In contrast to the BCR recognition, the
! 5!
TCR-pMHC interaction is extremely weak, having a half-life on the order of seconds.
As a result, staining rare antigen-specific T cells with pMHC monomers would not
survive the washing step in sample preparation for the flow cytometric analysis. (Davis
et al., 2011)
To improve the binding avidity for T cells, Davis and coworkers developed the
pMHC tetramer that can bind to multiple TCRs simultaneously. Specifically,
monomeric pMHC molecules were site-specifically biotinylated using the biotin protein
ligase BirA, and then conjugated to a fluorescently labeled streptavidin at the ratio of
4:1(Altman et al., 1996). Streptavidin is a homo-tetramer protein produced by the
bacterium Streptomyces avidinii.(Bayer et al., 1986) It has a molecular weight of 52
kDa and binds to 4 biotin molecules. The streptavidin-biotin binding is one of the
strongest non-covalent interactions, having an association constant (Kd) of 10
-15
M.
(Sano and Cantor, 1990) Therefore, it has been extensively used in a variety of
purification and detection strategies in biomedical and therapeutic studies. It was shown
that the streptavidin-derived pMHC tetramer was soluble and stable in solution, and
! 6!
that tetramers of HIV-derived peptides bound to human leucocyte antigen A2 (HLA-A2)
was capable of directly staining the HIV-specific CD8
+
cells. In addition, the number of
the cells stained with MHC tetramers correlated well with cytotoxicity assays. (Altman
et al., 1996)
Since the pMHC tetramer staining technique in 1996, it has been extended to a wide
range of applications in both fundamental and clinical immunological research. Below
are four representative applications: (1) It has been used to sensitively trace, selectively
isolate and purify T cells specific for a variety of microbial pathogens and
tumors.(Knabel et al., 2002) (2) The pMHC tetramers have also been used to determine
the dissociation rate of TCR.(Wang and Altman, 2003) (3) The pMHC tetramers have
been generated to verify or map disease-specific T cell antigens, which are important to
the development of vaccines and cancer immunotherapies.(Grotenbreg et al., 2008;
Newell et al., 2013) (4) The conjugation of auto-antigen derived pMHC tetramers and
toxin has the potential to be used to deplete auto-reactive T cells to treat autoimmune
diseases.(Dhodapkar et al., 2001) Clearly, the development of pMHC tetramers has
! 7!
provided an invaluable tool to the areas of T cell immunology and immunotherapy
development.
1.3. Alternatives to pMHC tetramers
Despite the enormous success of pMHC tetramers in staining and isolating
antigen-specific T cells, they still have major limitations. For example, pMHC
tetramers have been less successful in staining T cells with lower affinities, e.g., tumor-
and auto-reactive T cells which often have lower affinity for pMHCs than the typical
antivirus T cells. Further, the pMHC tetramer staining for low-affinity T cells is even
more problematic when used together with the CyTOF mass cytometer, a new variation
of flow cytometry that is capable of monitoring more than 40 markers simultaneously.
This is due to the extensive washing and the lengthy measurement associated with the
CyTOF analysis. Finally, the avidity of pMHC tetramers for T cells is still not sufficient
for in vivo applications.
To address these limitations, higher valency alternatives to pMHC tetramers have
! 8!
been developed, including pentamers, dextramers, quantum dots, and lipid
vesicles.(Davis et al., 2011) These multimeric complexes exhibited higher efficiency
than tetramers in staining antigen-specific T cells. For example, the dextramer was able
to detect autoimmune T cells with rather poor TCR affinity.(Dolton et al., 2014)
However, the dextramer, which consists of a dextran polymer carrying about 60
streptavidin and 180 pMHCs, has enhanced background staining. The other alternatives
are still relatively inefficient in detecting low-frequency, low-affinity T cells, and/or
inconvenient to produce.
Recently, Xie at al developed a novel photocrosslinkable pMHC which, even as a
monomer, can stain cognate T cells with high efficiency and excellent specificity.(Xie
et al., 2012) This reagent forms a covalent bond to the TCR, thus it does not dissociate
from the T cells and the staining signal lasts much longer than that of pMHC tetramers.
However, it is not amenable for in vivo applications due to the need of ultraviolet
irradiation at 365 nm.
In this study, we aim to design a novel pMHC multimer that can not only detect
! 9!
very low affinity T cells, but also have limited background staining.
1.4. DNA-directed assembly of proteins
We attempt to take advantage of the development of DNA self-assembly to design a
new type of pMHC multimers with higher valency and controllable shape.
Self-assembly of DNA nanostructures has allowed the construction of DNA-based
materials with well-define internal distance and shape, which makes it possible to
arrange different molecules in a controllable manner.(Chhabra et al., 2010; Rinker et al.,
2008) For instance, the Y-shaped DNA with three branched moieties has been used as
structural scaffold to assemble different functional architectures.
The principle of
designing of three oligonucleotide sequences was presented in Figure!2, for which the
first two oligonucleotides were partial complementary to form one arm and the third
oligonucleotide annealed with the rest of two oligonucleotides to form the other two
arms. The integral properties of the functional architectures mainly depend on the
configuration of the Y-shaped DNA scaffold and the length of each arm. With the short
! 10!
arms on Y-shaped DNA, the conformation can be stable below melting temperature and
the altered conformation heated beyond the melting temperature will also recover to
stable conformation after decreasing the temperature. Besides, Y-shaped DNA scaffold
also provides the angular flexibility of each arm to link to different
molecules.(Chatterjee et al., 2012) The Y-shaped DNA based materials have been used
for pathogen detection as well as for sustained release of siRNA drugs.(Roh et al., 2011)
In addition, the Y-shaped DNA containing cytosine-phosphate-guanine (CpG)
dinucleotide has been used to activate Toll-like receptors (TLRs) on dendritic cells, B
cells and macrophages, and stimulate these immune cells to produce
cytokines.(Matsuoka et al., 2010) Further, this immunostimulatory Y-shape DNA based
material has been successfully delivered to immune cells.(Mohri et al., 2015) Finally, a
series of Y-shaped DNAs with different sizes have been used to three anti-IgE
antibodies with tunable distances, and used subsequently to analyze the spatial
organization of IgE–FcεRI complexes in the mast cell signaling pathways.(Sil et al.,
2007)
! 11!
Our overall objective is to design DNA-presented pMHC multimers as next
generation T cell detection technique. As the first step to our objective, we will use the
trimeric Y-shaped DNA to present three streptavidin. This will be done by designing a
Y-shaped DNA with a biotin attached to the 5’-end of each of the three
oligonucleotides, followed by conjugation with three fluorescently-labeled streptavidin
molecules. Since each streptavidin still has the capacity to bind three additional biotin
molecules, this new scaffold would be able to present a total of nine pMHC complexes.
Compared with pMHC tetramers, our DNA-based pMHC nonamer has two potential
advantages: 1) it has increased valency; 2) the distance between pMHCs is tunable,
which may be important to the efficient binding to multiple TCRs simultaneously.
! 12!
2. Results and Discussion
2.1. Synthesis and characterization of Y-shaped DNA
To improve the avidity of pMHC tetramers, a Y-shaped DNA scaffold (Figure!2a)
designed to generate an improved multimeric complex, which can present nine pMHC
complexes of same origin. This Y-shaped DNA scaffold contains three branched moieties
assembled by annealing three complementary oligonucleotides. The 5’ terminus of these
oligonucleotides is commercially linked to one biotin molecule, so each arm of the
Y-shaped DNA can bind one fluorescencently-labeled streptavidin. Then the streptavidin
tetramer can subsequently bind three biotinylated MHC complexes. The final product
formed is a DNA scaffold containing three streptavidin molecules which each hold three
pMHC complexes, resulting in an entire pMHC nonamer. (Figure! 2b) One of the
advantages in utilizing a Y-shaped DNA scaffold to generate this pMHC complex is its
structural stability and angular flexibility. It has been demonstrated that the Y-shaped DNA
scaffold exerted maximum efficiency to construct a biological structure, given that the
length of each arm is 36 bases.(Sil et al., 2007)
! 13!
a. b.
Figure 2. A Y-shaped DNA and Y-shaped DNA scaffold based pMHC nonamer. a. The Y-shaped DNA
consists of three oligonucleotides. Each oligonucleotide has a biotin at its 5’-terminus. b. Schematic
illustration of the pMHC nonamer.
A similar design was also utilized to generate the Y-shaped DNA scaffold for this
pMHC multimeric complex. The entire Y-shaped DNA scaffold is 108 bases, and each
oligonucleotide used to generate the scaffold is 36 bases long. Each arm of the Y-shaped
DNA is 18 base pairs long, so all three arms of this scaffold contain a total of 54 base pairs.
In order to generate this Y-shaped DNA scaffold, equimolar concentrations of the three
oligonucleotides were mixed and annealed together under a programmable thermal cycle.
! 14!
The resulting synthesized Y-shaped DNA was characterized by a 3% agarose gel (Figure!3).
The annealing product was pure with only one thick and bright band in lane S. The
Y-shaped DNA contained 54 base pairs with 18 base pairs per arm, so this band shifted
faster than the 100 base pairs band in the ladder. Therefore, assembly of the Y-shaped DNA
was successful and with high yield. To extract and purify this product, the Y-shaped DNA
was recovered from the 3% agarose gel and precipitated with ethanol. The maximal yield of
recovery was 56.6%. The conformation of Y-shaped DNA remained stable when stored in
4°C.(Li et al., 2004) Since the Y-shaped DNA has been successfully prepared, this DNA
can be utilized as a scaffold to construct the pMHC nonamer.
Figure 3. Characterization of Y-shaped DNA.
The synthesized Y-shaped DNA was characterized on a 3%
agarose gel. Lane S represents the the annealed Y-shaped
DNA formed (108 base pairs) when the three 36-base pair
oligonucleotides are annealed together using a
programmable thermal cycle.
! 15!
2.2. Expression, in vitro refolding, and purification of streptavidin
Streptavidin is a 52 kDa homo-tetramer which can bind to four biotin molecules with
high affinity. Therefore, we took advantage of this property to construct the pMHC nonamer
by binding streptavidin irreversibly onto one biotin moiety present on the Y-shaped DNA
scaffold and onto three other biotinylated pMHC complexes.
1) Evaluation of commercial streptavidin
Streptavidin is known to dissociate to four 13 kDa subunits under high temperature
conditions. This homo-tetramer has also been found to form large molecular aggregates
under certain conditions, such as an acidic environment.(Bayer et al., 1986; Kurzban et al.,
1991) I first evaluated the properties of the homemade streptavidin and it turned out to be
the aggregates formed during refolding. The commercial streptavidin products were
subsequently supposed as the substitute for homemade streptavidin. The properties of
commercial streptavidin products were evaluated on 4%-12% Bis-Tris protein gels (Figure 4)
The four lanes labeled with 1# represent the commercial products from Sigma Aldrich
! 16!
dissolved in phosphate buffered saline (PBS) and stored in the -20°C. The last four lanes
labeled with 2# represent commercial streptavidin bought from AnaSpec, Inc, which was
freshly dissolved in PBS. Before running the samples in the Bis-Tris gel, the commercial
streptavidin products were mixed with 4X Bolt LDS sample buffer. Then these products
were either heated at 95°C for 10 minutes or unheated before running the samples through
the gel in order to compare the stability of the commercial streptavidin products using
heated conditions. Under the non-heated condition, both commercial streptavidin products
primarily formed aggregates, as shown by the presence of strong bands over 198 kDa
observed in lanes 2, 4, 6, and 8. On the other hand, some streptavidin tetramer bands at 52
kDa were observed in lanes 6 and 8, which correspond to the freshly dissolved commercial
product from AnaSpec, Inc. When these same streptavidin commercial products were
heated at 95°C for 10min, the streptavidin aggregates appeared to dissociate into monomeric
subunits, as observed by the presence of bands at 13 kDa in lanes 1, 3, 5, and 7. The results
of this gel are consistent with the fundamental properties previously mentioned above. In
summary, both commercial streptavidin products primarily consisted of aggregates. In
! 17!
addition, it appears that the proportion of active tetramer in commercial products decreases
after storage in -20°C since the portion of tetramer bands in the commercial streptavidin
from Sigma Aldrich was dramatically less than the second product. Streptavidin aggregation
is an issue for this research project because if aggregated, the protein may not specifically
bind one biotinylated site on the Y-shaped DNA and three pMHC’s to form the final pMHC
nonamer desired. Therefore, it is necessary to attain only the tetrameric streptavidin
conformation. Since the commercial streptavidin products appear to primarily yield
aggregated streptavidin, I next tried to express and prepare recombinant streptavidin using E.
coli.
Figure 4. Evaluation of two commercial streptavidin products on a
4%-12% Bis-Tris gel.!
1# represents the commercial streptavidin from Sigma Aldrich which was
stored in -20°C and 2# represents freshly dissolved streptavidin product
from AnaSpec, Inc. Each commercial product was treated with or without
heating at 95°C and with or without addition of reducing agent which was
applicable for the 4%-12% Bis-Tris gel. The bands over 198 kDa
demonstrate the presence of streptavidin aggregates. The bands at 52 kDa
represent tetrameric streptavidin. The bands at 13 kDa represent the
monovalent subunits. When both streptavidin commercial products were
heated, the bands at 198 kDa and 52 kDa disappeared, and one new band
at 13 kDa appeared Addition of reducing agent did not influence the
! 18!
results since there was no disulfide bond in commercial streptavidin products. M: marker. Heat+: Heat at 95°C
for 10 min. Heat-: Non-heated condition. Reducing +: 4X reducing agent was added to the samples. Reducing-:
No reducing agent was added.
2) Determination of inclusion body quality
In this project, the mutant streptavidin, which contains one cysteine at C’ terminus of
each subunit, was utilized for fluorescently labeling. The mutant streptavidin accumulates in
the inclusion body fraction of the bacterial cell lysate after the expression in BL21(DE3)
cells. The amino acid sequence of the mutant streptavidin subunit was
N’-MAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAP
ATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEA
NAWKSTLVGHDTFTKVGGSGCP-C’.
After the inclusion body is isolated, it is washed and then resuspended with 6M
guanidine hydrochloride, pH 1.5. Before the denatured streptavidin monovalent subunits are
refolded into active tetramers, the inclusion body purity has to be assessed since the
existence of impurities can potentially interfere with the refolding process. The denatured
inclusion bodies were diluted 25 times with the final concentration of 0.4 mg/mL and then
! 19!
evaluated using polyacrylamide gel electrophoresis (Figure! 5). The 13 kD subunits of
streptavidin predominated in the inclusion bodies with few impurities under both heated and
non-heated conditions. The pure and well-qualified inclusion bodies allowed for the
refolding of streptavidin.
Figure 5. Evaluation of the mutant streptavidin inclusion
body quality on a 4%-12% Bis-Tris gel.
The prepared inclusion bodies were denatured with 6mL 6M
guanidine hydrochloride, pH 1.5, and diluted 25 times with the
final concentration of 0.4 mg/mL for evaluation on the 4%-12%
Bis-Tris gel. Lane 1 represents the denatured streptavidin
inclusion body which only contained monovalent subunits at
around 13kDa under the non-heated condition. In lane 2, the
monovalent subunits remained stable after heating. M: marker;
Heat +: Heat at 95°C for 10 min; Heat -: No heating. SAV:
streptavidin
3) Refolding of streptavidin
! 20!
Streptavidin can be renatured directly after solubilization of the denatured inclusion
bodies. I adopted the quick dilution method to refold streptavidin, which involves rapidly
diluting the inclusion body protein—which is solubilized in a highly-concentrated
denaturing agent—into a rapidly mixing refolding buffer which contains little to no
denaturing agent. The specific details for refolding were initially based on the protocol from
Alice Y Ting et al. (Howarth and Ting, 2008). Up to 10mg of mutant streptavidin inclusion
body was slowly injected drop by drop into 250 mL of rapidly spinning refolding buffer.
Two different refolding buffers were tested: One containing 50 mM Tris pH 8.0, 150mM
NaCl, 2 mM EDTA and 1 mM TCEP (referred as Tris buffer) and the second containing
250 mL of PBS, 2 mM EDTA and 1 mM TCEP (referred as PBS buffer). The refolded
samples were concentrated and evaluated using polyacrylamide gel electrophoresis (Figure!
6).
The mutant streptavidin tetramer was 52 kDa with four subunits of 13 kDa. If the
monovalent streptavidin refolded into the 52 kDa tetramer, then what should be observed is
a band shifting larger than 49 kDa under the non-heated condition and the dissociation of
! 21!
this 52 kDa band into 13 kDa subunits after heating at 95 °C for 10 min. However, for both
Tris buffer and PBS buffer refolding systems, the results of refolding were quite similar.
Aggregates formed in the refolding systems as represented by bands over 198kDa in lanes 1
and 3 under the non-heated condition. These aggregate bands disassociated to monovalent
subunits, which is indicated by the 13 kDa bands in lanes 2 and 4. The results revealed that
the refolding of the denatured streptavidin monovalent subunits into tetrameric
conformation failed after following the procedures described above.
Figure 6. Detection of the refolded mutant streptavidin
on a 4%-12% Bis-Tris gel.
10 mg of denatured mutant streptavidin inclusion bodies
were diluted into 250 mL of either 50 mM Tris, pH 8.0, 150
mM NaCl, 2m M EDTA and 1 mM TCEP refolding buffer
or 250 mL of PBS, 2 mM EDTA and 1mM TCEP refolding
buffer, respectively. The refolded samples were either heated
at 95°C or unheated before detection on a 4%-12% Bis-Tris
gel. Under the non-heated condition, streptavidin aggregates
were present as observed by the bands over 198 kDa. These
aggregates decreased and resulted in the presence of 13 kDa
subunits upon heating. There was no significant difference in
the results between the two refolding buffers, for both did
not yield the desired streptavidin tetramer product at 52 kDa.
M: marker; Heat+: Heat at 95°C for 10 min; Heat -: No
! 22!
heating; PBS+: Refolded in the PBS refolding buffer; Tris+: Refolded in the Tris refolding buffer;
In order to address the problem, I have tried more than a dozen in vitro refolding
conditions. The entire troubleshooting process is listed in Table 1. The potential reasons for
the failure of streptavidin refolding are listed, followed by the alternative conditions I have
tested in an attempt to solve the issues. All of the conditions were mostly consistent with the
refolding procedure previously described except for a few alterations in certain trials.
! 23!
Table 1. Troubleshooting trials for refolding of streptavidin
Possible reasons Altered conditions
Low pH value of refolding buffer:
Since streptavidin has a mildly acidic isoelectric point
at 5.52, it is prone to aggregate at acidic or neutral
environments. A basic refolding buffer with higher pH
might contribute to stabilize the structure of the
tetramer.(Kurzban et al., 1991)
Instead of the Tris buffer at pH 8.0, I attempted
to use two different refolding buffers: the
50mM Tris pH 9.0, 150 mM NaCl, 2 mM
EDTA buffer and the 50 mM NaHCO
3
-Na
2
CO
3
pH 10.0, 150 mM NaCl, 2 mM EDTA buffer.
Large amount of inclusion bodies being injected:
The amount of inclusion bodies injected into the
refolding buffer might be too high to be diluted
completely. Therefore, streptavidin aggregates
subsequently form.
The amount of denatured streptavidin inclusion
body injected into the Tris refolding buffer is
decreased from 10 mg to 3 mg.
Concentration methods:
I suspected that the refolded tetramer aggregated on the
column of the Vivaspin concentrator (GE Healthcare)
during the concentration process.
The refolded samples were concentrated by
using both the Vivaspin concentrator as well as
ammonium sulfate ((NH
4
)
2
SO
4
) precipitation.
Both the concentrated and non-concentrated
samples were detected on the gel.
Aggregation during electrophoresis gel running:
The formation of intramolecular covalent bonds and
non-covalent bonds could potentially cause aggregation
during electrophoresis gel running.
The 4X reducing agent applicable for the
4%-12% Bis-Tris gel was mixed with the
refolded sample.
Cysteine residues interfering with the refolding:
The cysteine residues enable the formation of disulfide
bonds among subunits and interfere with the refolding.
The wild type streptavidin without cysteine
residues was obtained by replacing the
“GGSGCP” sequence at the C terminus of
mutant streptavidin to “KPSAAS” sequence.
! 24!
Unfortunately, none of the altered conditions listed in Table 1 resulted in the successful
refolding of streptavidin, for all of the 4%-12% Bis-Tris protein gel results indicated the
aggregation of streptavidin. At this point, I realized that the 4%-12% Bis-Tris protein gels
had the pH value of 6.4 and provided an acidic environment for the detected proteins.
Because streptavidin has a mildly acidic isoelectric point (pI) of 5.52, the acidity of the
Bis-Tris protein gel may induce streptavidin aggregation during the gel running step.
Considering this reason, I decided to evaluate the refolded samples and the streptavidin
commercial products on a 8%-12% Tris-Glycine protein gel, which has a pH value of 8.65
(Figure!7). For both the Tris and PBS refolding systems, no streptavidin aggregates over
198kDa were observed. Instead, streptavidin tetramers were present as observed by the
bands present at 52 kDa. These tetrameric bands also dissociated to 13 kDa monovalent
subunits upon heating at 95°C for 10 minutes, as can be seen in lanes 1, 3, 5, and 7. The
same results are observed with the streptavidin commercial products from AnaSpec. Inc.
Running the refolded streptavidin products on a Tris-glycine gel, which is manufactured at a
more basic pH, primarily contributed to the proper detection of the correctly-refolded
! 25!
tetramer. In later studies, streptavidin should be maintained in a basic environment (pH 8.0)
when utilized for conjugation to the Y-shaped DNA scaffold and to the biotinylated pMHC.
Figure 7. Detection of the refolded wild type streptavidin on a 8%-12% Tris-Glycine protein gel. 10mg
of denatured wild type streptavidin inclusion bodies were diluted into either the 250 mL of 50 mM Tris pH 8.0,
150 mM NaCl, 2 mM EDTA refolding buffer or the 250 mL of PBS, 2 mM EDTA refolding buffer,
respectively. The refolded samples were detected on a 8%-12% Tris-Glycine protein gel. The first four lanes
represent refolded protein from the Tris refolding buffer. Lanes 6, 7, 8, and 9 represent refolded protein from
the PBS refolding buffer. The last three lanes represent commercial streptavidin products, which were used as
positive controls. There were bands present at 52 kDa under non-heated conditions, demonstrating the
presence of streptavidin tetramer as shown in lanes 2, 4, 6, 8, 10, and 11. These 52 kDa bands disappeared
while 13 kDa bands appeared after heating at 95°C for 10 min, as shown in lanes 1, 3, 5, 7, and 9. This
observation indicates that under heating conditions, the 52 kDa streptavidin tetramer dissociates into 13 kDa
monovalent subunits, confirming the identity of the streptavidin tetramer. The bands present below the 52 kDa
streptavidin tetramer under both heated and non-heated conditions are impurities. M: marker; Heat+: Heat at
! 26!
95°C for 10 min; Heat-: No heating; PBS+: Refolded in the PBS refolding buffer; Tris+: Refolded in the Tris
refolding buffer;!
Since the streptavidin has been successfully re-natured, an established protocol can be
designed for the successful refolding of the denatured mutant streptavidin monomers into
tetramers. 10mg of inclusion body dissolved in 6M guanidine hydrochloride, pH 1.5, should
be diluted into 250 mL of rapidly spinning refolding buffer with 50 mM Tris pH 8.0, 150
mM NaCl, 2 mM EDTA and 1 mM EDTA drop by drop. Tris(2-carboxyethyl)phosphine
(TCEP), which is the reducing agent, is utilized to break disulfide bond between two
cysteine residues. Although refolding was successful in both Tris buffer and PBS buffer, the
potential advantages of the Tris buffer over PBS were that the Tris buffer has a stronger
buffer capability (due to its higher concentration) and that TCEP is more stable in the Tris
buffer. Then the refolded samples were concentrated with a Vivaspin concentrator and
evaluated on a 8%-12% Tris-Glycine protein gel, pH 8.65, under heated and non-heated
conditions.
! 27!
4) Purification of refolded streptavidin
The principle of the Superdex 200 10/300 GL column is based on gel filtration
chromatography, which separates proteins by size and molecular weight. Therefore, the
52kDa streptavidin tetramer should be well separated from the 13 kDa monovalent subunits
and 198kDa aggregates on the Superdex 200 10/300 column. To maintain the streptavidin
tetramer stability, the column running buffer was consistent with the refolding buffer, This
running buffer contains 50 mM Tris, pH 8.0, 150 mM NaCl, and 2 mM EDTA. The 52 kDa
streptavidin tetramer should be eluted at 15 mL from the Superdex 200 10/300 column.
However, this protein was observed to elute later at 23 mL (Figure!8). The streptavidin
tetramer may be eluting at a later volume because the protein may be interacting with the
beads in the Superdex 200 column. Therefore, I tried to increase pH value of the column
running buffer to pH 10, and I also added 10% glycerol to the running buffer. However, the
same eluting volume was retained, and the shape of the peak could not be improved. (data
not shown)
! 28!
Figure 8. Purification of refolded wild type streptavidin through the Superdex 200 10/300 GL column.
The refolded truncated wild type streptavidin was purified through the Superdex 200 10/300 GL column. The
streptavidin sample was run using a buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, and 2 mM EDTA.
The streptavidin peak eluted out at the fraction of 23.04 mL. The peaks at fractions 8.50 mL and 15.40 mL
were impurities.
To overcome the limitation brought by the extra interaction of streptavidin with the
Superdex 200 10/300 column, I decided to utilize the MonoQ column to purify streptavidin.
The MonoQ column is an anion exchange column and its principle is mainly based on the
separation of components based on the negative charge carried by molecules. The typical
program to purify proteins with anion-exchange involves binding the protein sample with a
low salt-containing buffer. Then the proteins are slowly eluted out of the column by
gradually increasing the salt concentration of the column running buffer. The isoelectric
! 29!
point (pI) of streptavidin is 5.52 so that it carries negative charge in the basic buffer. To
purify the refolded streptavidin, the protein was bound to the MonoQ column using a
running buffer that contained 25 mM Tris, pH 8.0. Then the wild type streptavidin was
gradually eluted with 25 mM Tris pH 8.0, 1M NaCl buffer. In Figure!9, the wild type
streptavidin eluted out with a sharp when the conductance was at 10mS/cm. The
streptavidin peak had high resolution which was clearly separated from the other peaks
which contained smaller impurities. Compared to the Superdex 200 column, purification of
streptavidin with the MonoQ column was shown to have higher resolution.
Figure 9. Purification of refolded wild type streptavidin through the MonoQ column.
The wild type streptavidin was bound to the MonoQ column with 25 mM Tris pH 8.0 binding buffer. The
streptavidin was gradually eluted out of the MonoQ under the gradient elution program with the gradual
increase in elution buffer concentration from 0% to 100% within 30 min. The streptavidin eluted at 6.63 mL
! 30!
when the conductance was at 10mS/cm. The peak at 8.31 mL consists of impurities.
!
!
5) Verification of the biotin-binding capability of homemade streptavidin
After the successful refolding and purification of streptavidin, I would need to verify if
it can bind biotin. I initially examined the conjugation between the biotinylated pMHC
molecule and the homemade streptavidin, but the result was complicated because the pMHC
was not fully biotinylated (data not shown). With respect to simplifying the test, I selected
the biotinylated ovalbumin to test the biotin-binding ability of streptavidin. The commercial
streptavidin, wild type streptavidin, and mutant streptavidin have all been utilized for the
conjugation tests. The streptavidin and biotinylated-ovalbumin were reacted at a molar ratio
of 10:1, respectively. Since ovalbumin has a molecular weight of 45 kDa, the biotinylated
ovalbumin and streptavidin complex should have a molecular weight of 97 kDa. When
compared to the control lane with only the biotinylated ovalbumin, the disappearance of the
ovalbumin band at 45 kDa and appearance of a new band at 100 kDa in lanes 8 and 11
under the non-heated condition was observed, indicating the successful conjugation of
biotinylated ovalbumin with streptavidin (Figure!10).
! 31!
Figure 10. Conjugation of biotinylated ovalbumin to streptavidin.
The biotinylated ovalbumin was conjugated to a 10 molar excess of commercial streptavidin products, wild
type streptavidin, and the mutant streptavidin, respectively. The reaction was conducted at room temperature
for 10 minutes. The conjugated samples were detected on a 8%-12% Tris-Glycine protein gel. The lane
“Non-Biotin OVA” corresponds to the non-biotinylated ovalbumin. The lane “Biotin OVA” corresponds to the
biotinylated ovalbumin. The lanes for “SAV commercial product” represent the conjugation products between
biotinylated ovalbumin and commercial streptavidin. The lanes for “SAV WT” and “SAV-1C-live” represent
conjugation products between biotinylated ovalbumin and wild type streptavidin and mutant streptavidin,
respectively. The ovalbumin is a 45 kDa protein, so the biotinylated ovalbumin and streptavidin complex is 97
kDa. When compared to the control lane with only biotinylated ovalbumin, the disappearance of the
ovalbumin band at 45 kDa and appearance of a new band at 100 kDa under the non-heated condition indicated
the successful conjugation of ovalbumin with streptavidin, as shown in lanes 8 and 11. M: marker; Heat+:
Heat at 95°C for 10 min; Heat -: No heating; Ovalbumin+: Add ovalbumin; Ovalbumin-: No ovalbumin
! 32!
2.3. Conjugation reaction
Before construction of pMHC nonamers, the binding properties of streptavidin to
biotin-linked oligonucleotides and biotinylated protein should first be verified.!
Generally, the three biotins moieties present on each branch of the Y-shaped DNA
should occupy one binding site on streptavidin separately, leaving the rest of the nine
biotin-binding sites from each streptavidin tetramer for binding biotinylated pMHC
complexes. However, the three biotin molecules on the Y-shaped DNA have the possibility
to crosslink between streptavidin without control, even with a starting molar ratio of 1:3
between the Y-shaped DNA and streptavidin. Considering this possible situation, another
alternative to construct the Y-shaped DNA bound to three streptavidin tetramers is to
conjugate streptavidin with the three oligonucleotides separately before subsequently
annealing the conjugation products together.
Although the two starting materials can react at equimolar concentrations, streptavidin
can additionally conjugate to oligonucleotides at molar ratios of 1:0, 1:1, 1:2, 1:3 and 1:4.
The MonoQ column was applied to collect the streptavidin conjugated with only one
! 33!
biotinylated oligonucleotide (Figure!11.!Conjugation!of!biotinylated!oligonucleotide!to!
streptavidin., the first peak at the elution volume of 9.22 mL was s considered as the
desired conjugation product based on two reasons: (1) The UV 254 and UV 280 absorption
peaks completely overlapped, which demonstrates the elution of DNA and protein
simultaneously. (2) Streptavidin conjugated to one oligonucleotide would carry less
negative charges than if it were conjugated to two, three or four oligonucleotides. For all of
the three individual oligonucleotides utilized to generate the Y-shaped DNA scaffold, the
oligonucleotide-streptavidin conjugation products were generated to produce the Y-shaped
DNA.
! 34!
Figure 11. Conjugation of biotinylated oligonucleotide to streptavidin.
The 1# oligonucleotide with biotin at the 5’ end was conjugated to streptavidin at a molar ratio of 1:1. The
reaction was conducted at room temperature for 10 min. Then the conjugated products were detected through
the MonoQ column. 25 mM Tris pH 8.0 buffer was used as the binding buffer, and 25 mM Tris pH 8.0, 1 M
NaCl was utilized as the elution buffer. The conjugation products were gradually eluted out of the MonoQ
under the gradient elution program with elution buffer increasing from 0% to 100% within 30min. DNA was
monitored at an absorbance of 254 nm, and the protein was monitored at an absorbance of 280 nm.
! 35!
2.4. Future plan
So far, streptavidin has been prepared with efficient biotin-binding ability as well as the
streptavidin-oligonucleotide conjugation products. The availability of these materials allows
us to continue constructing the pMHC nonamers. The mutant streptavidin with 4 cysteine
residues at the C terminus, has also been conjugated to each of the individual
oligonucleotides making up the Y-shaped DNA scaffold. The next step with this mutant
streptavidin will involve labeling the protein with fluorescein-5-maleimide. Subsequently,
the three fluorescein-streptavidin-oligonucleotide conjugation products will be annealed to
form the Y-shaped DNA scaffold. Once the scaffold is obtained, nine refolded p-MHC
complexes can be loaded onto the Y-shaped DNA scaffold. Therefore, the pMHC nonamers
will be constructed in this way. One important application of these pMHC nonamers is to
improve the sensitivity of detecting antigen-specific T cells, especially for low affinity
T-cell populations, compared to tetramers. In a later study, cell staining by flow cytometry
will be adopted to evaluate the detection of TCRs on antigen-specific T cells. Our hope is
that this novel DNA-streptavidin multimer will enable the presentation of a variety of
! 36!
pMHC class I and II molecules, of both human and mouse origin, for the successful
detection of low-affinity T cells in cancer and autoimmune diseases.
! 37!
3. Method
3.1. Synthesis and characterization of Y-shaped DNA
Materials
The building blocks for the Y-shaped DNA includes three commercially synthesized
oligonucleotides of equal length. Their sequences are listed below:
#1 5’-Biotin-ACCACTGGATCCGCATGACATTCGCCGTAAGCACAC-3’
#2 5’-Biotin-GTGTGCTTACGGCGAATGACCGAATCAGCCTGCTGA-3’
#3 5’-Biotin-TCAGCAGGCTGATTCGGT TCA TGCGGATCCAGTGGT-3’
Construction and purification of Y-shaped DNA
The three commercially-synthesized oligonucleotides were mixed at equimolar
concentrations of 20 µM and were annealed to form the Y-shaped DNA following the steps
below: (1) denature at 95°C for 2 min; (2) gradual cool down to 70°C at a rate of 1°C per
minute; (3) incubation at 70°C for 5 min; (4) gradual cool down to 50°C for annealing with
a continuous temperature decrease at the a rate of 0.1°C per minute. The annealed Y-shaped
! 38!
DNA was characterized by a 3% agarose gel.
To extract and purify the Y-shaped DNA, the DNA in the 3% agarose gel was first run
onto a double-layer membrane consisting of glass fiber and dialysis tubing membranes.
Then the DNA product was recovered from the double-layer membrane through
centrifugation and solubilization in TBE buffer. The Y-shaped DNA was subsequently
purified by ethanol precipitation. The Y-shaped DNA was stable when stored at -20°C.
3.2. Expression, in vitro refolding and purification of streptavidin
Materials
The amino acid sequence of the mutant streptavidin subunit was
N’-MAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAP
ATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEA
NAWKSTLVGHDTFTKVGGSGCP-C’, with one cysteine at the C terminus. The wild type
streptavidin was obtained by replacing the “GGSGCP” sequence in the mutant subunit with
“KPSAAS” through molecular cloning.
! 39!
Expression and preparation of streptavidin inclusion body
A 1L culture of BL21(DE3) E.coli cells transformed with streptavidin plasmids were
incubated at 37°C, 225 rpm until the OD
600nm
reached 0.6. 1 mL of 1 M IPTG was added to
the 1L of culture to induce the expression. After 4h, all cell pellets were collected from the
1L of culture and re-suspended into 5 mL lysis buffer plus 5 mM PMSF, 5 mM DTT, 5mg
lysozyme, 50 µg/mL DNAseI, 50µM MgCl
2
, 0.5 µg/mL leupeptin and 5 µg/mL pepstatin.
The suspended cell pellets were maintained at room temperature for 1h and subsequently
disrupted with sonication to obtain inclusion bodies. After sonication, the inclusion bodies
were washed 2 times with detergent buffer (1% sodium deoxycholate, 1% NP-40, 50 mM
Tris pH8.0, 200 mM NaCl, 1 mM EDTA), 3 times with Wash buffer 1 (5% Triton X-100,
50 mM Tris pH 8.0, 100 mM NaCl, 0.1% azide, 1 mM EDTA) and 2 times with Wash
buffer 2 (50 mM Tris pH8.0, 100 mM NaCl, 0.1% azide, 1 mM EDTA). For every round of
washing, the inclusion bodies were required to be completely dissolved in the buffer and
! 40!
recovered after centrifugation at 4°C, 13000rpm for 10min. The washed inclusion bodies
were stored at -20°C.
Streptavidin refolding
We adopted the dilution method for refolding based on Mark Howarth et al., (Howarth
and Ting, 2008), where 10 mg of dissolved inclusion bodies were slowly diluted to 250 mL
of refolding buffer. The inclusion bodies were solubilized with 6 mL 6M guanidine
hydrochloride (GuHCl), pH 1.5, followed with centrifugation at 20000g, 4°C for 30 min.
The resulting supernatant was carefully added drop by drop into the rapidly spinning
refolding buffer. The refolded samples were concentrated with a Vivaspin concentrator and
stored at 4°C without stirring. The refolded streptavidin was evaluated using SDS-PAGE.
More details of the refolding procedure have been discussed in the results.
Purification of streptavidin
To test the quality of streptavidin, the refolded streptavidin should be purified before its
! 41!
conjugation to biotinylated oligonucleotides and proteins. The first method to purify
streptavidin was by fast protein liquid chromatography (FPLC) with the Superdex 200
10/300 column. The components of the running buffer were consistent with the refolding
buffer. The other method was to use the MonoQ column (anion exchange column). The
binding buffer used contains 25 mM Tris-HCl, pH 8.0, and the elution buffer contained 25
mM Tris-HCl, pH 8.0, and 1 M NaCl. The streptavidin was eluted using a gradient elution
program with the composition of elution buffer increasing from 0% to 100% within 30min.
3.3. Conjugation reaction
Conjugation of biotinylated oligonucleotide to streptavidin
The purified streptavidin and oligonucleotide with biotin at the 5’ terminus were mixed
at equimolar concentrations to set up a 50µM reaction system. The reaction was kept at
room temperature for 10min and evaluated by the MonoQ column. Both A
280nm
and A
254nm
were monitored during the elution step.
! 42!
Conjugation of biotinylated ovalbumin to streptavidin
The biotinylated ovalbumin was produced in our lab. The biotinylated ovalbumin and
streptavidin were mixed at molar ratios of 1:10. The reaction was conducted at room
temperature for 10min and evaluated by SDS-PAGE.
! 43!
References
Altman, J.D., Moss, P.A., Goulder, P.J., Barouch, D.H., McHeyzer-Williams, M.G., Bell,
J.I., McMichael, A.J., and Davis, M.M. (1996). Phenotypic analysis of antigen-specific T
lymphocytes. Science 274, 94-96.
Arstila, T.P., Casrouge, A., Baron, V., Even, J., Kanellopoulos, J., and Kourilsky, P. (1999).
A direct estimate of the human αβ T cell receptor diversity. Science 286, 958-961.
Bayer, E.A., Ben-Hur, H., Gitlin, G., and Wilchek, M. (1986). An improved method for the
single-step purification of streptavidin. Journal of biochemical and biophysical methods 13,
103-112.
Chatterjee, S., Lee, J.B., Valappil, N.V., Luo, D., and Menon, V.M. (2012). Probing
Y-shaped DNA structure with time-resolved FRET. Nanoscale 4, 1568-1571.
Chhabra, R., Sharma, J., Liu, Y., Rinker, S., and Yan, H. (2010). DNA self-assembly for
nanomedicine. Advanced drug delivery reviews 62, 617-625.
Davis, M.M., Altman, J.D., and Newell, E.W. (2011). Interrogating the repertoire:
broadening the scope of peptide–MHC multimer analysis. Nature Reviews Immunology 11,
! 44!
551-558.
Davis, M.M., and Bjorkman, P.J. (1988). T-cell antigen receptor genes and T-cell
recognition.
Dhodapkar, M.V., Steinman, R.M., Krasovsky, J., Munz, C., and Bhardwaj, N. (2001).
Antigen-specific inhibition of effector T cell function in humans after injection of immature
dendritic cells. The Journal of experimental medicine 193, 233-238.
Dolton, G., Lissina, A., Skowera, A., Ladell, K., Tungatt, K., Jones, E., Kronenberg Versteeg, D., Akpovwa, H., Pentier, J., and Holland, C. (2014). Comparison of peptide major histocompatibility complex tetramers and dextramers for the identification of
antigen specific T cells. Clinical & Experimental Immunology 177, 47-63.
Grotenbreg, G.M., Roan, N.R., Guillen, E., Meijers, R., Wang, J.-h., Bell, G.W., Starnbach,
M.N., and Ploegh, H.L. (2008). Discovery of CD8+ T cell epitopes in Chlamydia
trachomatis infection through use of caged class I MHC tetramers. Proceedings of the
National Academy of Sciences 105, 3831-3836.
Howarth, M., and Ting, A.Y. (2008). Imaging proteins in live mammalian cells with biotin
! 45!
ligase and monovalent streptavidin. Nature protocols 3, 534-545.
Huppa, J.B., Axmann, M., Mörtelmaier, M.A., Lillemeier, B.F., Newell, E.W.,
Brameshuber, M., Klein, L.O., Schütz, G.J., and Davis, M.M. (2010). TCR–peptide–MHC
interactions in situ show accelerated kinetics and increased affinity. Nature 463, 963-967.
Knabel, M., Franz, T.J., Schiemann, M., Wulf, A., Villmow, B., Schmidt, B., Bernhard, H.,
Wagner, H., and Busch, D.H. (2002). Reversible MHC multimer staining for functional
isolation of T-cell populations and effective adoptive transfer. Nature medicine 8, 631-637.
Kurzban, G., Bayer, E., Wilchek, M., and Horowitz, P. (1991). The quaternary structure of
streptavidin in urea. Journal of Biological Chemistry 266, 14470-14477.
Li, Y., Tseng, Y.D., Kwon, S.Y., d'Espaux, L., Bunch, J.S., McEuen, P.L., and Luo, D.
(2004). Controlled assembly of dendrimer-like DNA. Nature materials 3, 38-42.
Lillemeier, B. (2010). TCR and LAT occur in separate membrane domains and concatenate
during activation. Nature Immunol 11, 90-96.
Matsui, K., Boniface, J.J., Steffner, P., Reay, P.A., and Davis, M.M. (1994). Kinetics of
T-cell receptor binding to peptide/I-Ek complexes: correlation of the dissociation rate with
! 46!
T-cell responsiveness. Proceedings of the National Academy of Sciences 91, 12862-12866.
Matsuoka, N., Nishikawa, M., Mohri, K., Rattanakiat, S., and Takakura, Y. (2010).
Structural and immunostimulatory properties of Y-shaped DNA consisting of
phosphodiester and phosphorothioate oligodeoxynucleotides. Journal of Controlled Release
148, 311-316.
McHeyzer-Williams, M.G., Nossal, G., and Lalor, P.A. (1991). Molecular characterization
of single memory B cells. Nature 350, 502-505.
Mohri, K., Kusuki, E., Ohtsuki, S., Takahashi, N., Endo, M., Hidaka, K., Sugiyama, H.,
Takahashi, Y., Takakura, Y., and Nishikawa, M. (2015). Self-assembling DNA dendrimer
for effective delivery of immunostimulatory CpG DNA to immune cells.
Biomacromolecules 16, 1095-1101.
Newell, E.W., Sigal, N., Nair, N., Kidd, B.A., Greenberg, H.B., and Davis, M.M. (2013).
Combinatorial tetramer staining and mass cytometry analysis facilitate T-cell epitope
mapping and characterization. Nature biotechnology 31, 623-629.
Rinker, S., Ke, Y., Liu, Y., Chhabra, R., and Yan, H. (2008). Self-assembled DNA
! 47!
nanostructures for distance-dependent multivalent ligand–protein binding. Nature
nanotechnology 3, 418-422.
Roh, Y.H., Ruiz, R.C., Peng, S., Lee, J.B., and Luo, D. (2011). Engineering DNA-based
functional materials. Chemical Society Reviews 40, 5730-5744.
Sano, T., and Cantor, C.R. (1990). Expression of a cloned streptavidin gene in Escherichia
coli. Proceedings of the National Academy of Sciences 87, 142-146.
Sil, D., Lee, J.B., Luo, D., Holowka, D., and Baird, B. (2007). Trivalent ligands with rigid
DNA spacers reveal structural requirements for IgE receptor signaling in RBL mast cells.
ACS chemical biology 2, 674-684.
VALITUTTI, S., MULLER, S., CELLA, M., PADOVAN, E., and LANZAVECCHIA, A.
(1995). SERIAL TRIGGERING OF MANY T-CELL RECEPTORS BY A FEW
PEPTIDE-MHC COMPLEXES. Nature 375, 148-151.
Wang, X.L., and Altman, J.D. (2003). Caveats in the design of MHC class I
tetramer/antigen-specific T lymphocytes dissociation assays. Journal of immunological
methods 280, 25-35.
! 48!
Xie, J., Huppa, J.B., Newell, E.W., Huang, J., Ebert, P.J., Li, Q.-J., and Davis, M.M. (2012).
Photocrosslinkable pMHC monomers stain T cells specifically and cause ligand-bound
TCRs to be'preferentially'transported to the cSMAC. Nature immunology 13, 674-680.
Abstract (if available)
Abstract
T cells, which play a central role in cellular immunity, express distinct T cell receptors (TCR) to recognize antigenic peptides bound to major histocompatibility complex molecules (MHCs). The ability to detect, quantify, isolate, and characterize often very rare antigen-specific T cells from a population is very important to both fundamental and clinical immunology. Monomeric peptide-MHC (pMHC) molecules cannot stably stain cognate T cells because the TCR–pMHC interaction has a low affinity (KD=1-100 μM) and a fast disassociation half-life (t1/2 = 1-10 s). This problem has been significantly addressed by the development of pMHC tetramers and other forms of multimers, which are capable of binding to multiple TCRs simultaneously. However, it has been known that some T cells, e.g., cancer or auto-reactive T cells, can express extremely low-affinity TCRs that are beyond the detection limit of pMHC tetramers. In this thesis, we aim to develop a novel type of multimeric pMHC reagent that has significantly enhanced detection sensitivity. Specifically, we attempt to use a Y-shaped DNA scaffold to present three streptavidin molecules, which in turn will be conjugated to multiple biotinylated pMHC molecules. The DNA-directed assembly of pMHC complexes will have two main characteristics: 1) it is capable of presenting nine pMHCs in one complex
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Asset Metadata
Creator
Fu, Ting
(author)
Core Title
Towards DNA-directed assembly of pMHC multimers for detection of low-affinity T cells
School
School of Pharmacy
Degree
Master of Science
Degree Program
Molecular Pharmacology and Toxicology
Publication Date
04/19/2016
Defense Date
03/25/2016
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
low affinity,OAI-PMH Harvest,pMHC,streptavidin,T cell receptor,Y-shaped DNA
Format
application/pdf
(imt)
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Xie, Jianming (
committee chair
), Okamoto, Curtis (
committee member
), Stiles, Bangyan (
committee member
)
Creator Email
tingfu@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c40-233315
Unique identifier
UC11278038
Identifier
etd-FuTing-4297.pdf (filename),usctheses-c40-233315 (legacy record id)
Legacy Identifier
etd-FuTing-4297.pdf
Dmrecord
233315
Document Type
Thesis
Format
application/pdf (imt)
Rights
Fu, Ting
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Access Conditions
The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...
Repository Name
University of Southern California Digital Library
Repository Location
USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA
Tags
low affinity
pMHC
streptavidin
T cell receptor
Y-shaped DNA