Close
About
FAQ
Home
Collections
Login
USC Login
Register
0
Selected
Invert selection
Deselect all
Deselect all
Click here to refresh results
Click here to refresh results
USC
/
Digital Library
/
University of Southern California Dissertations and Theses
/
Enhancing the anti-HIV potency of eCD4-Ig by unnatural amino acid mutagenesis
(USC Thesis Other)
Enhancing the anti-HIV potency of eCD4-Ig by unnatural amino acid mutagenesis
PDF
Download
Share
Open document
Flip pages
Contact Us
Contact Us
Copy asset link
Request this asset
Transcript (if available)
Content
Enhancing the Anti-HIV Potency of eCD4-Ig by Unnatural
Amino Acid Mutagenesis
by
MANYUN CHEN
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfilment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)
August 2017
I
Table of contents
Acknowledgements ................................................................................................... III
List of figures ............................................................................................................. IV
Abbreviation .............................................................................................................. VI
Abstract ................................................................................................................... VIII
1. Introduction .............................................................................................................. 1
1.1 AIDS and HIV entry process ................................................................................................. 1
1.2 Development of eCD4-Ig as a potent HIV entry inhibitor .................................................... 2
1.3 Rationale of this project......................................................................................................... 6
1.3.1 Structural analysis of CD4 binding HIV gp120 ............................................................. 6
1.3.2 Incorporation of unnatural amino acids into recombinant proteins .............................. 10
1.3.3 Protein-protein fusion catalyzed by sortase A .............................................................. 10
1.4 Overall Objectives ............................................................................................................... 11
2. Materials and Methods .......................................................................................... 14
2.1 Equipment and reagents ...................................................................................................... 14
2.2 Experiment materials ........................................................................................................... 14
2.2.1 Bacterial strains for cloning and expression ................................................................. 14
2.2.2 Expression vectors ........................................................................................................ 14
2.3 Methods ............................................................................................................................... 17
2.3.1 Construction of recombinant DNA............................................................................... 17
2.3.2 Expression of recombinant proteins in E. coli .............................................................. 20
2.3.3 Ni-NTA affinity chromatography ................................................................................. 21
2.3.4 Ion-exchange chromatography ..................................................................................... 22
2.3.5 Gel filtration chromatography ...................................................................................... 23
2.3.6 Protein A affinity chromatography ............................................................................... 23
3. Site-Specific Incorporation of Unnatural Amino Acids into CD4 (D1-D2) ...... 25
3.1 Expression, refolding and purification of the wild-type CD4 (D1-D2) ............................... 25
3.2 Expression, refolding and purification of UAA incorporated CD4 (D1-D2) ...................... 30
II
3.2.1 BipAla incorporated CD4 (D1-D2) .............................................................................. 30
3.2.2 IodoF incorporated CD4 (D1-D2) ................................................................................ 33
3.3 Summary ............................................................................................................................. 37
4. Sortase-Catalyzed Conjugation of CD4 (D1-D2) and IgG1-mim2 .................... 38
4.1 Preliminary study of CD4 (D1-D2)–sfGFP assembly ......................................................... 38
4.1.1 Expression and purification of GB1-SortA-His6 ......................................................... 38
4.1.2 Expression and purification of G5-sfGFP .................................................................... 42
4.1.3 CD4 (D1-D2)-sfGFP assembly under different conditions .......................................... 45
4.1.4 Verification of CD4 (D1-D2)-sfGFP fusion product.................................................... 47
4.2 CD4 (D1-D2)–IgG1-mim2 assembly catalyzed by sortase A ............................................. 49
4.2.1 Expression and purification of G5-IgG1-mim2 ............................................................ 49
4.2.2 Expression and purification of SortA ........................................................................... 52
4.2.3 Fusion reaction of CD4 (D1-D2) and G5-IgG1-mim2 ................................................. 55
4.3 Summary ............................................................................................................................. 57
5. Conclusion .............................................................................................................. 58
6. References ............................................................................................................... 59
III
Acknowledgements
I hereby would like to express my heartfelt gratitude to my advisor, Dr. Jianming Xie,
for the offering me the opportunity to work on this research project and for his guidance
during my study in the University of Southern California. I enjoyed the research experience
in his laboratory, from which I learned a lot about protein engineering and immunotherapy.
I also would like to thank my committee members, Dr. Curtis T. Okamoto and Dr.
Wei-Chiang Shen for their suggestions on this thesis and for their encouragement and help
during my graduate study.
Moreover, I would love to thank Dr. Liang Rong for his guidance in the cloning and
protein work and Rebecca Lim for her help in the cell culture work. They also provided me
with useful suggestions on this thesis. I additionally would like to thank the previous lab
member Yiao Wang, who worked together with me on the early construction of
recombinant DNA. Also, I want to thank all the other lab members for their assistance and
support.
IV
List of figures
Figure 1. Overview of HIV life cycle and entry process.
Figure 2. Comparison of eCD4-Ig variants and HIV-1 neutralizing antibodies.
Figure 3. Overall structure of CD4 binding HIV gp120.
Figure 4. Comparison of CD4 binding HIV gp120 and CD4 binding MHC-II.
Figure 5. Scheme for the genetic incorporation of unnatural amino acids.
Figure 6. Diagram of the construction of CD4 (D1-D2)-IgG1-mim2 with the
incorporation of unnatural amino acid.
Figure 7. Plasmid maps of the expression vectors.
Figure 8. Construction of pET22b-CD4(1-177)-LPETG-His6.
Figure 9. Purification of wild-type CD4 (D1-D2) by Ni-NTA affinity chromatography.
Figure 10. Purification of wild-type CD4 (D1-D2) by gel filtration.
Figure 11. Purification of BipAla-incorporated CD4 (D1-D2) by Ni-NTA affinity
chromatography.
Figure 12. Expression of IodoF-incorporated CD4 (D1-D2).
Figure 13. Purification of IodoF-incorporated CD4 (D1-D2) by Ni-NTA affinity
chromatography.
Figure 14. Purification of IodoF-incorporated CD4 (D1-D2) by gel filtration.
Figure 15. Purification of GB1-SortA-His6 by Ni-NTA affinity chromatography.
Figure 16. Purification of GB1-SortA-His6 by ion-exchange chromatography.
V
Figure 17. Purification of G5-sfGFP by Ni-NTA affinity chromatography and ion-
exchange chromatography.
Figure 18. Purification of G5-sfGFP by gel filtration.
Figure 19. CD4 (D1-D2)-sfGFP protein assembly catalyzed by sortase A.
Figure 20. Verification and separation of CD4 (D1-D2)-sfGFP assembly product by ion-
exchange chromatography.
Figure 21. Expression of G5-IgG1-mim2 and protein purification by Protein A affinity
chromatography.
Figure 22. Purification of G5-IgG1-mim2 by gel filtration.
Figure 23. Purification of His6-SUMO-SortA by Ni-NTA chromatography.
Figure 24. Purification of SortA by Ni-NTA chromatography and gel filtration.
Figure 25. SDS-PAGE analysis of CD4 (D1-D2)-IgG1-mim2 fusion reaction catalyzed
by sortase A.
VI
Abbreviation
aaRS, aminoacyl-tRNA-synthetase
AA V , adeno-associated virus
AIDS, acquired immunodeficiency syndrome
BipAla, biphenylalanine
bNAb, broadly neutralizing antibody
CCR5, C-C chemokine receptor type 5
CD4bs, CD4 binding site
CD4 (D1-D2), CD4 domains 1 and 2
CXCR4, C-X-C chemokine receptor type 4
DTT, Dithiothreitol
E. coli, Escherichia coli
Env, HIV envelope glycoprotein
sfGFP, superfolder green fluorescent protein
HIV , human immunodeficiency virus
IodoF, Iodo-phenylalanine
IPTG, Isopropyl β-D-1-thiogalactopyranoside
mAb, monoclonal antibody
mRNA, Messenger RNA
NAb, neutralizing antibody
VII
PBS, Phosphate Buffered Saline
PCR, polymerase chain reaction
Phe, phenylalanine
SortA, Sortase A
SDS-PAGE, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
tRNA, Transfer RNA
UAA, unnatural amino acid
VIII
Abstract
Human immunodeficiency virus (HIV) entry into the host cell is initiated by the
sequential binding of the viral envelope protein gp120 to the host cell’s receptor CD4 and
co-receptor CCR5 or CXCR4. Recently Dr. Farzan’s group developed a novel HIV entry
inhibitor eCD4-Ig by fusing a CD4 fragment and a small CCR5-mimetic sulfopeptide to
the N- and C-termini of an IgG Fc fragment. It was shown that eCD4-Ig was able to bind
avidly and cooperatively to gp120 and that its anti-HIV breadth and potency were better
than the currently characterized broadly neutralizing antibodies to HIV-1. The research
goal of our project was to further enhance the anti-HIV potency of eCD4-Ig by unnatural
amino acid mutagenesis. Based on the crystal structure of CD4-gp120 complex, Phe43 of
CD4 is surrounded by a large hydrophobic cavity of gp120. Therefore, we propose to
mutate Phe43 to a larger unnatural amino acid such as biphenylalnine (BipAla) and iodo-
phenylalanine (IodoF), which can potentially enhance the binding between eCD4-Ig and
gp120. To achieve this objective, we combined two novel protein chemistry techniques:
one is the site-specific incorporation of unnatural amino acids into recombinant proteins,
the other is the sortase-catalyzed protein-protein conjugation. So far I have successfully
expressed CD4 (D1-D2) variants containing BipAla or IodoF in E. coli, and expressed the
IgG1 Fc fragment fused with CCR5mim2 sulfopeptide mim2 (IgG1-mim2) in mammalian
cells. I have also successfully used sortase to conjugate CD4 (D1-D2) and IgG1-mim2,
although the yield is still low. In future work, it is necessary to further optimize the
IX
conjugation between CD4 (D1-D2) and IgG1-mim2, and measure the binding affinity of
the resulting eCD4-Ig variants for gp120.
Key words: CD4, HIV gp120, unnatural amino acid, sortase A.
1
1. Introduction
1.1 AIDS and HIV entry process
Human immune deficiency virus (HIV) is a retrovirus that attacks the immune system
and causes acquired immune deficiency syndrome (AIDS). Since its discovery in 1981
(Friedman-Kien et al., 1981; Gallo et al., 1982), the HIV/AIDS epidemic has become an
unprecedented global health crisis. It is estimated that more than 70 million people have
been infected, over 35 million have already died of AIDS, and about 2 million are newly
infected per year (UNAIDS, 2015). A great deal of effort has been made to develop
antiretroviral molecules that target the different stages in the HIV lifecycle, including viral
attachment, fusion, reverse transcription, integration, and maturation (Figure 1a, Laskey
and Siliciano, 2014). However, as of now there is still no cure for HIV.
As the first step in infection, HIV entry is an important target for the development of
anti-HIV therapeutics. Shortly after the identification of HIV-1, it was found that HIV
selectively infects CD4
+
T cells (Klatzmann et al., 1984) and that monoclonal CD4
antibodies can inhibit HIV infection in vitro (Dalgleish et al., 1984). These findings led to
the recognition of CD4 as the primary receptor for HIV . Further investigation demonstrated
that HIV entry also requires one of the following two chemokine receptors: CXCR4 (Feng
et al., 1996) and CCR5 (Cocchi et al., 1995; Choe et al., 1996). On the viral side, the
envelope glycoprotein (Env) involved in HIV entry is a trimer of heterodimers formed by
the surface subunit gp120 and the transmembrane subunit gp41 (Wyatt et al., 1998). As
shown in Figure 1b (Wilen et al., 2012), the entry of HIV into host cells begins with the
binding of gp120 to CD4. The CD4-binding site is located at the interface of the outer and
inner domains of gp120 (Kwong et al., 1998). The CD4 binding induces organization of
the envelope glycoprotein trimeric spikes, resulting in an outward rotation of each gp120
monomer and rearrangement of the gp41 region to expose the binding site for chemokine
receptors CCR5 or CXCR4 (Zhang et al., 1999). This sequential binding of CD4 and
2
CCR5/CXCR4 allows HIV to attach and fuse with target cells and to deliver the genetic
payload into the host cell.
1.2 Development of eCD4-Ig as a potent HIV entry inhibitor
Tremendous effort has been made to develop neutralizing antibodies (NAbs) capable
of blocking the interaction between HIV and the host cell; however, the extraordinary
genetic diversity of HIV has been a major barrier. Specifically, it was found that the HIV-
1 envelope proteins from different strains can differ in more than 30% of their amino acid
sequences (Barouch, 2008; Gaschen et al., 2002). While multiple broadly NAbs (bNAbs)
have been developed which can bind and neutralize diverse HIV strains, an estimated 10-
50% of HIV-1 isolates are still partially or wholly resistant to the currently available bNAbs
(Gardner et al., 2015).
Inspired by the interaction between CD4 and gp120 in the HIV entry process,
researchers have also attempted to use CD4 and its derivatives as HIV entry inhibitors. In
a previous research, soluble CD4 protein (sCD4) demonstrated anti-HIV activity in vitro
(Hussey et al., 1988); however, early-stage clinical trials failed to achieve antiviral activity,
most likely because sCD4 has a short plasma half-life and its affinity for gp120 is low
(Daar et al., 1990). Thereafter, a variety of CD4 derivatives have been developed in order
to further enhance the anti-HIV potency. For example, CD4-immunoglobulin (CD4-Ig)
hybrid molecules, in which the D1 and D2 domains of CD4 are attached to the Fc fragment
of IgG, have been extensively studied as an anti-HIV entry candidate (Fletcher et al., 2007).
CD4-Ig has a ten fold longer plasma half-life than sCD4, and a broader virus neutralization
capability than any bNabs. However, it was found that at low concentrations, both sCD4
and CD4-Ig can enhance HIV-1 infection because their binding to gp120 can induce
conformational changes of gp120 and thus facilitate the association of the virus envelope
glycoprotein and target cell surface coreceptors CCR5 or CXCR4. To address this
limitation, Dr. Farzan and colleagues recently developed a series of fusion proteins between
3
CD4-Ig and CCR5 mimetic sulfopeptides, referred to as eCD4-Ig, which can target both
the CD4 and CCR5 binding sites of HIV envelope glycoprotein (Figure 2a) (Gardner et al.,
2015). They showed that these eCD4-Ig variants were more potent and broader than the
best bNAbs, as the eCD4-Ig variants could neutralize the HIV isolates that were resistant
to the neutralizing antibodies and their geometric mean IC50 values are lower than that of
the bNAbs (Figure 2b, 2c). The most potent eCD4-Ig variant, eCD4-Ig
mim2
, differs from
eCD4-Ig by a single Ala to Tyr substitution in the CCR5 mimetic peptide and has an IC50
of about 1.5 μg/ml or less for all the tested HIV isolates. Finally, they showed that fully
functional rhesus eCD4-Ig could be expressed in vivo for more than 40 weeks using an
adeno-associated virus (AAV) vector and serve as an alternative to HIV vaccines.
4
Figure 1. Overview of HIV life cycle and entry process. (a) Scheme of stages in HIV
life cycle that are targeted by anti-HIV drugs (Laskey and Siliciano, 2014). (b) Scheme of
the HIV entry process: HIV Env first binds to CD4 on the host cell surface; this binding
causes conformational changes in Env, allowing binding to the coreceptor; in the next step,
the fusion peptide of gp41 inserts into the target membrane, followed by the formation of
a six-helix bundle and the viral and cell membrane fusion (Wilen et al., 2012).
a
b
5
Figure 2. Comparison of eCD4-Ig variants and HIV-1 neutralizing antibodies. (a)
Structure of eCD4-Ig. CD4-Ig is comprised of CD4 domains 1 and 2 (blue) fused to the
human IgG1 Fc domain (grey). In eCD4-Ig, the sulfopeptide CCR5mim1 (red) is fused to
the C terminus of CD4-Ig. The sequence of the CCR5 N terminus is provided for
comparison. Common residues, including four CCR5 sulfotyrosines, are shown in red.
CCR5mim1 Ala 4 (blue) is substituted with Tyr in CCR5mim2. (b) HIV-1 pseudotyped
with the SIVmac239 Env was incubated with varying concentrations of CD4-Ig, eCD4-Ig
variants or CD4bs antibodies, and the percentages of infection were plotted. (c)
Comparison of the IC50 values of CD4-Ig, eCD4-Ig variants or CD4bs antibodies in
different HIV-1, HIV-2 and SIV isolates (Gardner et al., 2014).
a
b
c
6
1.3 Rationale of this project
1.3.1 Structural analysis of CD4 binding HIV gp120
The research goal of this thesis project is to devise a rational design strategy to further
enhance the anti-HIV potency of eCD4-Ig. Based on the X-ray crystal structure of the CD4-
gp120 complex, the residue Phe43 in the CD4 domain D1 sits inside the gp120 binding
pocket (Figure 3, Kwong et al., 1998) and accounts for more than 23% of the total
intermolecular contacts. However, Phe43 is surrounded by a large hydrophobic cavity of
gp120, termed “Phe43 cavity”. We hypothesize that substitution of a larger unnatural
amino acid for Phe43 can help to fill the Phe43 cavity and enhance the binding affinity
between the resulting eCD4-Ig mutant and gp120.
Previous studies have designed therapeutic molecules to target the Phe43 cavity of
gp120. For example, a small molecule binding to the Phe43 cavity was developed to block
CD4-gp120 interaction (Tintori et al., 2013). Additionally, a 27-mer peptide, CD4M33,
which contains a large aromatic amino acid biphenylalanine (BipAla) at the position
corresponding to Phe43 of CD4, was shown to bind gp120 with an affinity approximately
10-fold higher than that of the phenylalanine derivative (Stricher et al., 2008). Finally, a
series of mutant CD4 domain 1 (mD1.2), in which Phe43 was mutated to unnatural amino
acids such as BipAla and iodo-phenylalanine (IodoF), were produced by using chemically
aminoacylated tRNAs and a cell-free translation system. These BipAla- and IodoF-
containing mutants demonstrated enhanced binding to gp120 compared to the wild-type
CD4 fragment (Yu et al., 2014). Combined together, these findings have provided a solid
base for our hypothesis that an enhanced eCD4-Ig can be produced by insertion of a large
unnatural amino acid to fit the large hydrophobic binding pocket of gp120.
Furthermore, we attempt to examine whether the incorporated unnatural amino acid
can reduce the binding between eCD4-Ig and MHC class II, a potential adverse effect
associated with these types of CD4-containing immunoadhesins for treating HIV infection.
7
MHC class II molecules are expressed on the surface of antigen-presenting cells such as
dendritic cells, macrophages, and B cells, and they present antigenic peptides to induce
CD4
+
helper T cell responses. MHC II is also the natural substrate of CD4, and CD4 recruit
Lck (a protein tyrosine kinase) to regulate T-cell activation. Therefore, eCD4-Ig and other
soluble CD4 derivatives can potentially suppress CD4
+
T-cell immunity. Previous research
on the CD4 protein structure has illuminated that its Phe43 residue is involved in binding
to the hydrophobic pockets of both gp120 and MHC-II (Zhou et al., 2007). However, there
are two main differences between the Phe43-binding pockets of gp120 and of MHC II.
First, the pocket of gp120 is larger than that of MHC II. Second, the pocket of gp120 is
mostly hydrophobic but also has two charged residues (D368 and E370) close to the
entrance. On the contrary, the pocket of MHC II is completely hydrophobic (Figure 4a and
4b). Based on these structural differences, we hypothesize that it may be possible to
identify an unnatural amino acid that can enhance the binding of eCD4-Ig for gp120 but
minimize its binding for MHC-II.
We plan to combine two novel protein chemistry techniques to produce unnatural
amino acid-containing eCD4-Ig. First, we will use the E. coli protein expression system to
incorporate unnatural amino acids into human CD4 fragments. Second, we will use the
sortase A-catalyzed protein-protein conjugation method to fuse the resulting CD4 mutant
and the Fc fragment of IgG.
8
Figure 3. Overall structure of CD4 binding HIV gp120. The Phe43 sits inside the
binding pocket of CD4 binding HIV gp120. The ribbon diagram shows gp120 (blue) in
complex with CD4 (light blue) and gp120 antibody Fab 17b in light blue (light chain) and
purple (heavy chain). The side chain of Phe 43 (yellow) on CD4 is shown. (Kwong et al.,
1998)
9
Figure 4. Comparison of CD4 binding HIV gp120 and CD4 binding MHC-II. (a)
Phe43 binding pocket of CD4 binding HIV gp120. (b) Amino-acid residues near the Phe43
binding pocket of CD4 binding HIV gp120. (c) Phe43 binding pocket of CD4 binding
MHC-II. (d) Amino-acid residues near the Phe43 binding pocket of CD4 binding MHC-II.
(e) Structures of BipAla and IodoF.
e
d
c
b
a
10
1.3.2 Incorporation of unnatural amino acids into recombinant proteins
A recently developed method has made it possible to site-specifically incorporate a
variety of unnatural amino acids into recombinant proteins in E. coli (Xie and Schultz,
2006). Specifically, a unique stop codon TAG (amber codon) and an orthogonal
aminoacyl-tRNA-synthetase (aaRS)/tRNA pair were used for the incorporation of the
unnatural amino acid with high fidelity and efficiency. Based on the consideration that the
new components added should be orthogonal, not cross-reactive, to the endogenous
components in E. coli, the specific aaRS/tRNA pair was chosen from Methanococcus
jannaschii (Wang et al., 2000). The selective Methanococcus jannaschii TyrRS recognizes
both the suppressor tRNA and the unnatural amino acid, which is specifically added onto
the 3’ end of tRNA. The suppressor tRNA carrying the unnatural amino acid is able to
recognize the stop codon, UAG, on the mRNA and deliver the unnatural amino acid at that
site. Meanwhile, the releasing factor 1 (RF1) in E. coli, which also recognizes the stop
codon, can compete with the amber-suppressor tRNA and stop protein synthesis, thus
generating a truncated protein. The truncated protein can be separated from the full length
protein through affinity chromatography purification or gel filtration.
1.3.3 Protein-protein fusion catalyzed by sortase A
The Staphylococcal aureus sortase A is a bacterial transpeptidase that can covalently
attach proteins to the bacterial cell wall. Specifically, sortase A cleaves between threonine
and glycine at an LPXTG recognition motif to generate an acyl-enzyme intermediate,
which then reacts with an N-terminal glycine to generate a native amide bond (Mazmanian
et al., 1999). This chemistry has been increasingly exploited for site-specific ligation
between a protein displaying the C-terminal LPETGX motif to a second protein containing
an N-terminal multi-glycine motif (Mao et al., 2004). The robust and facile nature of this
reaction makes sortase A an ideal enzyme for in vitro protein ligation (Levary et al., 2011).
11
1.4 Overall Objectives
This project aims to construct the eCD4-Ig with incorporation of BipAla or IodoF at
the Phe43 site to further enhance the anti-HIV potency of eCD4-Ig. Figure 6 shows the
diagram of the project design. Specifically, the CD4 domains 1 and 2 (D1-D2) with the
incorporated unnatural amino acid will be expressed in E. coli, and the IgG1 Fc fragment
fused with the CCR5mim2 sulfopeptede (IgG1-mim2) will be expressed in mammalian
cells. To allow subsequent protein-protein conjugation under the catalysis of sortase A, the
CD4 (D1-D2) variants will be constructed with a C-terminal LPETG-His6 motif, and the
IgG fragment will contain an N-terminal G5 motif. Sortase A will then be used to catalyze
the conjugation between CD4 (D1-D2) and IgG1-mim2.
12
Figure 5. Scheme for the genetic incorporation of unnatural amino acids (Kim, Axup
and Schultz, 2013).
13
Figure 6. Diagram of the construction of CD4 (D1-D2)-IgG1-mim2 with the
incorporation of unnatural amino acid.
14
2. Materials and Methods
2.1 Equipment and reagents
The UV-Vis spectrophotometer Nanodrop 2000 was purchased from Thermo
Scientific (Rockford, IL). The T100 Thermal Cycler PCR machine was obtained from Bio-
Rad (Hercules, CA). The ÄKTA Protein Purification System was purchased from GE
Healthcare (Chicago, IL). HisTrap HP Ni-NTA column, Mono Q 5/50 GL, Mono S 5/50
GL, HiTrap Q FF column, Superdex 200 Increase 10/300 GL SEC column and HiTrap
Protein A HP column were purchased from GE Healthcare (Chicago, IL). Tris-glycine
SDS-PAGE gels were purchased from Invitrogen (Waltham, MA) and GenScript
(Pascataway, NJ). The other consumable materials used in molecular biology experiments
were purchased from companies including QIAGEN (Valencia, CA), Agilent
Technologies (Santa Clara, CA) and NEB (Ipswich, MA).
2.2 Experiment materials
2.2.1 Bacterial strains for cloning and expression
The E.coli DH5α competent cells were purchased from Invitrogen (Waltham, MA)
and the E.coli BL21 (DE3) competent cells were purchased from NEB (Ipswich, MA).
DH5α competent cells were used for subcloning of genes into plasmid vectors. BL21 (DE3)
was a T7 expression strain used for high efficiency protein expression.
2.2.2 Expression vectors
In this research, pET22b was used for bacterial expression of CD4, GFP and SortA.
The pET expression system is the most commonly used system for cloning and expression
of recombinant protein in E.coli.
The pEVOL system is a newly developed system for the expression of the specific
aminoacyl tRNA synthetase for incorporation of an unnatural amino acid (Young et al.,
2010). In this research, pEVOL-BipRS and pEVOL-IodoFRS plasmids were constructed
15
for the incorporation of BipAla and IodoF, respectively. The BipRS and IodoFRS genes
were amplified from the pBK plasmids by PCR.
The CMVR vector was used for the expression of the IgG1-mim2 fragment in
mammalian cells.
The pET22b(+), pEVOL-aaRS and CMVR maps are shown in Figure 7. All the
vectors were preserved in our lab.
16
Figure 7. Plasmid maps of the expression vectors. (a) pET-22b(+) map. (b) pEVOL-
aaRS map (Young et al., 2010). (c) CMVR- eCD4-Ig plasmid map (Gardner et al., 2015).
a
b
c
17
2.3 Methods
2.3.1 Construction of recombinant DNA
2.3.1.1 Gene amplification
All the target genes for plasmid construction were amplified by PCR using Pfx DNA
polymerase. The reaction system contained 0.3 μM each primer, 0.3 mM dNTPs, 1× Pfx
Buffer, 1 mM MgSO4, and 1.25 units Pfx DNA polymerase. The following protocol
provides the typical set-up for the PCR reaction (the annealing temperature is determined
by primer Tm):
95 ° C 2 min
95 ° C 30 s
35 cycles 55 ° C 30 s
68 °C 1 min for each 1000 bp
68 °C 5 min
4 ° C ∞
After the PCR reaction, the PCR products were analyzed by agarose gel
electrophoresis and the amplified genes were recovered by the Gel DNA Recovery Kit
(Zymo Research).
2.3.1.2 Double digestion with restriction enzymes
The double digestion was performed according to the NEB standard restriction
enzyme double digest protocol. The reaction system contained 1× CutSmart Buffer, NEB
restriction enzymes and DNA substrate. The reagents were incubated at 37 ° C for 2-4h.
The double-digested PCR products were recovered by the DNA clean kit. The double-
digested vector was analyzed by agarose gel electrophoresis and recovered by the Gel DNA
Recovery Kit.
2.3.1.3 Ligation of gene insert and linear vector
The T4 ligation system was prepared according to the NEB T4 ligation protocol. The
ratio of linear vector and double-digested PCR product was set as 1:7. The reagents were
18
incubated at room temperature for 1 h or 16 ° C overnight.
2. 3.1.4 Transformation of E. coli
The heat-shock method was used for bacterial transformation according to the
following protocol. E. coli competent cells (-80 °C) were thawed on ice. DNA (1 μL) was
added to the competent cells (25 μL) in a 1.5mL tube. The mixture was incubated on ice
for 30 min, and then heated in a 42° C water bath for 30s. After an incubation on ice for 2
min, 800 μL SOC (without antibiotic) was added to each tube, and the cells were recovered
at 37° C in a shaking incubator for 1h. Cells were centrifuged at 6000 rpm for 1min, and
then resuspended in 200 μL media. Cells were then plated onto a 10 cm LB agar plate
containing the appropriate antibiotic, and incubated at 37 ° C overnight. Positive colonies
were selected by colony PCR and verified by DNA sequencing (GENEWIZ, Inc.).
2.3.1.5 Construction of the expression vector of CD4 (D1-D2)
pET-22b was used as the expression vector for CD4. Figure 8a shows the scheme for
CD4 expression vector. Primers were designed according to the upstream and downstream
of sequences of CD4 (Figure 8b). The DNA fragment of CD4 (D1-D2) was amplified by
PCR from the CD4 (1-177) in CMVR-eCD4-Ig with a peptide “LPETGHHHHHH” added
to the C-terminus. Both the PCR product and the pET-22b vector were double digested by
Nde I and Xho I. After ligation with T4 DNA ligase and transformation into DH5α
competent cells, positive colonies were selected for sequencing to get the pET22b-CD4(1-
177)-LPETG-His6 plasmid.
In order to incorporate unnatural amino acids into the 43th site of CD4 domain D1,
the codon for Phe43 (TTC) was mutated to the amber codon (TAG) using the
QuickChange® Site-Directed Mutagenesis kit (Agilent Inc.).
19
10 20 30 40 50 60
KKVVLGKKGD TVELTCTASQ KKSIQFHWKN SNQIKILGNQ GSFLTKGPSK LNDRADSRRS
70 80 90 100 110 120
LWDQGNFPLI IKNLKIEDSD TYICEVEDQK EEVQLLVFGL TANSDTHLLQ GQSLTLTLES
130 140 150 160 170 180
PPGSSPSVQC RSPRGKNIQG GKTLSVSQLE LQDSGTWTCT VLQNQKKVEF KIDIVVLAFQ
190 200 210 220 230 240
KASSIVYKKE GEQVEFSFPL AFTVEKLTGS GELWWQAERA SSSKSWITFD LKNKEVSVKR
250 260 270 280 290 300
VTQDPKLQMG KKLPLHLTLP QALPQYAGSG NLTLALEAKT GKLHQEVNLV VMRATQLQKN
310 320 330 340 350 360
LTCEVWGPTS PKLMLSLKLE NKEAKVSKRE KAVWVLNPEA GMWQCLLSDS GQVLLESNIK
370 380 390 400 410 420
VLPTWSTPVQ PMALIVLGGV AGLLLFIGLG IFFCVRCRHR RRQAERMSQI KRLLSEKKTC
430
QCPHRFQKTC SPI
Figure 7. Construction of pET22b-CD4(1-177)-LPETG-His6. (a) Scheme of the
expression vector for wild-type CD4. (b) The amino acid sequence of full length CD4. The
sequence for CD4 (D1-D2) is labelled in yellow and the Phe43 is labelled in red. (c)
Structure of the CD4 extracellular domains (D1-D4) (generated by PymoL based on Figure
3), and the corresponding amino acid sequence for the CD4 domains.
a
b
c
20
2.3.1.6 Construction of the expression vector of aminoacyl-tRNA synthetases
Two copies of aminoacyl-tRNA synthetase gene sequences were inserted into the
pEVOL plasmid, because the protein expression level is low if the expression vector only
contains one copy of the gene. One of the tRNA synthetase genes is expressed utilizing an
endogenous promoter, while the other synthetase gene is regulated by the arabinose operon
system.
The BipRS(D286R) and IodoFRS genes were subcloned from the pbk-BipRS (D286R)
and pbk-IodoFRS plasmids. To insert the first copy of aaRS into the pEVOL vector, both
the PCR product and the vector were double digested by Bgl II and Sal I. After T4 ligation
and transformation into DH5α competent cells, positive colonies were sent for sequencing
to get the pEVOL-BipRSI or pEVOL-IodoFRSI plasmid. Then the successfully
constructed pEVOL-aaRSI plasmids were further digested by Nde I and Pst I to insert the
second copy of aaRS. The procedure was the same as that of the first insertion. The
sequence of pEVOL-BipRSI-BipRSII or pEVOL-IodoFRSI-IodoFRSII plasmids were
confirmed by GENEWIZ, CA, USA.
2.3.2 Expression of recombinant proteins in E. coli
The plasmid for expression was transformed into E. coli BL21 (DE3). The isolated
colony was transferred to 25 ml of LB media with appropriate antibiotics and incubated at
37° C, 225 rpm until the OD600 reaches 0.6. The starter culture was added into 1 L LB with
antibiotics and incubated for 1-4 hours until OD600 reaches 0.6. IPTG was added to a final
concentration of 1 mM to induce protein expression. For expression of inclusion bodies,
the bacterial culture was shaken for 4 hours at 37° C. For expression of soluble protein, the
bacterial culture was incubated at 18° C overnight after IPTG induction. After protein
expression was completed, all cell pellets were collected from the 1L culture by
centrifugation.
The cell pellets were re-suspended into 30 mL lysis buffer (containing 1 mM PMSF,
21
1 mM DTT, 30 mg lysozyme, 50 µ g/mL DNAseI, 10 µ M MgCl2, 0.5 µ g/mL leupeptin and
5 µ g/mL pepstatin). The suspended cell pellets were incubated on ice for 1 h and
subsequently disrupted by sonication.
After sonication, the suspension was centrifuged at 4 ° C, 13000 rpm for 20 minutes.
For soluble proteins (GFP and SortA in this research), the supernatant was collected. For
the protein expressed in the form of inclusion bodies (CD4 in this research), 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 pH8.0, 100 mM NaCl, 0.1% sodium azide, 1 mM EDTA) and 2 times
with Wash Buffer 2 (50 mM Tris pH8.0, 100 mMNaCl, 0.1% sodium azide, 1 mM EDTA).
For every round of washing, the inclusion bodies were completely dissolved in the buffer
and recovered after centrifugation at 4 ° C, 13000 rpm for at least 10 min.
2.3.3 Ni-NTA affinity chromatography
Histidine is an α-amino acid that contains an imidazole side chain. The electrophilic
imidazole moiety can form ligand-interaction with a group of transition metal ions
including Cu
2+
, Zn
2+
, Ni
2+
, Co
2+
and Fe
3+
. The Ni-NTA affinity chromatography is
developed based on the high affinity and selectivity of Ni-NTA agarose for recombinant
fusion proteins containing poly-histidine. The 6× His tag (His6) is the DNA sequence
specifying a string of six histidine residues in the expression vector which results in a 6× His
peptide fused on the N- or C-terminus of the protein. His6 is neutral at pH8.0 and non-
immunogenic, and it has merely subtle effect on protein expression, refolding and function.
The following protocol for Ni-NTA affinity chromatography was used in this research
(normal condition):
Equilibration: the constant-flow pump was washed with ddH2O at 10 mL/min for 2 min;
the Ni-NTA column was connected to the pump and equilibrated with 25 mL normal
binding buffer (20 mM Tris pH 8.0, 250 mM NaCl, 25 mM Imidazole) at 2 mL/min.
22
Sample application: the protein sample was loaded at 0.5 mL/min; the column was
washed with 25 mL binding buffer at 2 mL/min to remove the impurities.
Elution: the A pump of the ÄKTA system was placed into normal binding buffer and B
pump was placed into normal elution buffer (20 mM Tris pH 8.0, 250 mM NaCl, 500 mM
Imidazole); the system was washed with 100% B until the baseline was stable; the system
was washed with 0% B until the new baseline is stable; the system parameters were set
(alarm: 0.5 MPa, flow rate: 0.5 mL/min, monitor: UV280 and UV260); the Ni-NTA column
was connected and equilibrated until the baseline was stable; the elution program was
performed (autozero UV; flow rate: 2 mL/min, gradient: 0-100% B in 10 min, fraction: 2
mL/tube); the column was washed into ddH2O.
The fractions with high UV280 were collected and verified by SDS-PAGE.
2.3.4 Ion-exchange chromatography
Ion-exchange chromatography separates molecules based on ionic interactions
between the molecules and the ionizable functional group in the stationary phase of the
column. The soluble molecules in the mobile phase undergo electrostatic interactions with
opposite charges on the stationary phase matrix, and the molecules can be separated
because of different charged groups. The composition of the binding buffer and elution
buffer is decided by the isoelectric point of the protein. Hyposaline solution is commonly
used as the binding buffer and hypersaline solution is commonly used as the elution buffer.
Mono Q 5/50 GL and Mono S 5/50 GL columns were used in this research according to
the following protocol:
Equilibration: the A pump of the ÄKTA system was placed into binding buffer and B
pump was placed into normal elution buffer; the system was washed with 100% B until the
baseline was stable and then the system was washed with 0% B until the new baseline was
stable; the system parameters were set (alarm: 2 MPa, flow rate: 0.5 mL/min, B%: 0,
monitor: UV280 and UV260); The column was connected and equilibrated until the baseline
23
was stable.
Sample application: the injection loop was washed with 3 times the loop volume of
binding buffer; the sample was injected.
Elution: the elution program was performed (e.g. flow rate: 0.5 mL/min, gradient: 0-100%
B in 20mL, fraction: 0.25 mL/tube); wash the column into ddH2O. Note: The program is
different for different proteins.
The fractions with high UV280 were collected and verified by SDS-PAGE.
2.3.5 Gel filtration chromatography
Gel filtration chromatography separates molecules based on the molecular size. The
porous beads in the stationary phase result in a faster migration rate of larger molecules
and slower migration rate of smaller molecules. Superdex column (matrix composition:
cross-linked agarose and dextran) is widely used for its high-resolution with short run times
and good recovery. In this research, Superdex 200 Increase 10/300 GL SEC column was
used according to the following protocol:
Equilibration: the A pump of the ÄKTA system was placed into 1× PBS and B pump was
placed into ddH2O; the column was equilibrated with 40 mL 1× PBS.
Sample application: the injection loop was washed with 3 times the loop volume of
1× PBS; the sample was centrifuged at 13000 rpm for 5 min to remove any possible
precipitates; the sample was injected.
Elution: the system parameters were set (alarm pressure: 2 MPa; flow rate: 0.5 mL/min,
elution volume: 35 mL; fraction: 2mL/tube); the column was washed into ddH2O.
The fractions with high UV280 were collected and the protein size was calculated based on
the elution volume. The protein sample was verified by SDS-PAGE.
2.3.6 Protein A affinity chromatography
Staphylococcal protein A binds all IgG molecules of subclasses 1, 2 and 4 with high
selectivity. This molecule has been widely utilized to purify antibodies (Hober et al., 2007).
24
In this research, HiTrap Protein A HP column was used according to the following protocol:
Equilibration sample application: the constant-flow pump was washed with ddH2O at 10
mL/min for 2 min; the Protein A column was connected to the pump and equilibrated with
15 mL binding buffer (1× PBS) at 1 mL/min.
Sample application: the protein sample was loaded at 0.5 mL/min; the column was
washed with 10 mL binding buffer at 1 mL/min to remove the impurities.
Elution: the A pump of the ÄKTA system was placed into binding buffer and B pump was
placed into elution buffer (e.g., 0.1 M citric acid, 150 mM NaCl, pH=3.0); the system was
washed with 100% B until the baseline was stable and then the system was washed with
0% B until the new baseline was stable; the system parameters were set (alarm: 0.5 MPa,
flow rate: 1 mL/min, monitor: UV280 and UV260); Protein A column was connected and
equilibrated until the baseline was stable; the elution program was performed (flow rate: 1
mL/min, 100% B, fraction: 0.5 mL/tube); the column was washed into ddH2O. 1 mL 1M
Tris (pH=8.0) was added into each fraction collection tubes ahead of time to prevent protein
degradation.
The fractions with high UV280 were collected and the protein samples were verified by
SDS-PAGE.
25
3. Site-Specific Incorporation of Unnatural Amino Acids into CD4 (D1-
D2)
The HIV entry inhibitor eCD4-Ig is a fusion protein of the following three fragments:
1) the human CD4 domains D1-D2; 2) the IgG1 Fc domain; and 3) a C-terminal
sulfopeptide mim2 which is derived from the chemokine receptor CCR5. Previous work
showed that in the CD4-gp120 complex, the residue Phe43 of CD4 is surrounded by a large
hydrophobic cavity of gp120, termed “Phe43 cavity”. Our hypothesis is that replacement
of Phe43 with a large unnatural amino acid, such as p-iodo-L-phenylalanine, can fill the
Phe43 cavity and enhance the binding between eCD4-Ig and gp120. To produce this eCD4-
Ig mutant, we have devised a novel three-step strategy: 1) express p-iodo-L-phenylalanine
containing human CD4 proteins in E. coli using the technique of site-specific unnatural
amino acid incorporation, 2) express the IgG1-mim2 fusion protein in 293T cells, and 3)
conjugate the mutant CD4 and the IgG1-mim2 proteins by the sortase-catalyzed reaction.
3.1 Expression, refolding and purification of the wild-type CD4 (D1-D2)
Before attempting to incorporate unnatural amino acids into CD4 (D1-D2), we first
examined whether the wild-type protein could be expressed in E. coli. CD4 (D1-D2) is
composed of 177 amino acids and has a molecular weight of 20.9 kDa (Figure 7b). To
enable sortase A-catalyzed ligation between CD4 (D1-D2) and IgG1-mim2, we added an
LPETG-His6 tag to the C-terminus of CD4 (D1-D2). We constructed the expression vector
pET-22b-CD4(1-177)-LPETG-His6, and expressed the protein as inclusion bodies in E.
coli BL21 (DE3). Around 50 mg inclusion bodies could be obtained from 1 L LB culture.
Next, we attempted to refold CD4(1-177)-LPETG-His6 in vitro. There are two pairs
of cysteine residues in CD4 (D1-D2), and the disulfide bond can be formed in either D1 or
D2 during the refolding process. As a result, there are three different redox states of CD4
(D1-D2). It was previously reported that gp120 was incapable of binding fully oxidized or
26
fully reduced CD4 (Cerutti et al., 2014). To achieve high CD4-gp120 affinity, the majority
of the refolded CD4 (D1-D2) should contain only on disulfide bond. In this research, CD4
(D1-D2) was refolded by the dilution method into the refolding buffer (0.1 mM GSSG, 1
mM GSH, 0.4 M L-Arginine, 10% sucrose, 50 mM Na2CO3, pH=9.6). After 48 h refolding,
CD4 (D1-D2) was dialyzed into Ni-NTA binding buffer and purified by the Ni-NTA
column. The protein was eluted out as the concentration of imidazole increased (Figure 9a).
Monomer CD4 migrated between 15 and 20 kDa. Some CD4 dimers could be visualized
on the SDS-PAGE gel (Figure 9b).
27
Figure 9. Purification of wild-type CD4 (D1-D2) by Ni-NTA affinity chromatography.
(a) Chromatogram of wild-type CD4 (D1-D2) purified by Ni-NTA column. (b) SDS-
PAGE gel analysis of protein sample after Ni-NTA purification.
a
b
28
In order to remove the dimers and to attain purified monomer CD4 (D1-D2), the
protein was further purified by gel filtration using the Superdex 200 Increase 10/300 GL
SEC column. According to the gel filtration chromatogram, the protein was eluted out at
around 17 mL and there was a peak on the left shoulder (Figure 10a). From the SDS-PAGE
analysis, we found that the CD4 (D1-D2) dimers were eluted out earlier than the monomer
protein, and we collected fractions (35, 38) containing pure CD4 (1-177)-LPETG-His6
(Figure 10b).
It was previously reported that CD4 (D1-D2) in different redox states migrated at
different position on the SDS-PAGE gel, and the oxidized CD4 migrated faster than the
reduced CD4 (Cerutti et al., 2014). We found that the majority of the purified CD4 (D1-
D2) monomer migrated around 19kDa with a smaller minor band around 17kDa (Figure
10b), but when the fraction 36 of the gel-filtration-purified CD4 monomer was treated with
DTT, the disulfide bond was reduced and the protein was in the fully reduced state
migrating at around 24 kDa. Thus, it was confirmed that the majority of the refolded CD4
(D1-D2) was in an intermediate state containing only one disulfide bond which was the
desired redox state of CD4 for gp120 binding.
After refolding and purification, we were able to obtain around 1 mg refolded CD4
(D1-D2) from 12 mg inclusion body, so the total yield of wild-type CD4 (D1-D2)
expression and purification was around 4 mg refolded protein from 1 L LB culture.
29
Figure 10. Purification of wild-type CD4 (D1-D2) by gel filtration. (a) Chromatogram
of wild-type CD4 (D1-D2) purified by Superdex 200 10/300 GL SEC column. (b) SDS-
PAGE gel analysis of protein sample after gel filtration.
a
b
30
3.2 Expression, refolding and purification of UAA incorporated CD4 (D1-D2)
To replace the Phe43 in CD4 (D1-D2) with the unnatural amino acids, an amber
nonsense mutation (TTC to TAG) was introduced in the pET22b-CD4(1-177)-LPEG-His6
plasmid. This amber nonsense codon can be recognized by amber suppressor tRNA that
carries unnatural amino acids. We inserted genes encoding biphenylalanyl-tRNA
synthetase (BipAlaRS) and iodo-phenylalanyl-tRNA synthetase (IodoFRS) into pEVOL
vector containing the amber suppressor tRNA gene, respectively. (See Section 2.3.1.6 for
the methods)
The full length CD4 (D1-D2) with the incorporation of unnatural amino acid is around
20.9 kDa, while the truncated protein containing only the first 42 amino acids in CD4
domain 1 is less than 5 kDa. The His6 tag is constructed on the C-terminus of CD4 (D1-
D2) and the truncated protein has no His6 tag. Therefore, only the full length CD4 (D1-D2)
with UAA incorporation can bind the Ni-NTA column.
3.2.1 BipAla incorporated CD4 (D1-D2)
In order to further enhance the affinity of BipRS with amber suppressor tRNAs, a
D286R mutation was introduced into the BipRS genes. The Asp286 in the aaRS is the key
residue for specific recognition of the first base of the anticodon. However, in the wild-
type BipRS, Asp286 is too small to interact specifically with the residue at position34 of
amber suppressor tRNA. Therefore, we replaced Asp286 with a larger amino acid, arginine.
The expression vectors pET22b-CD4(1-177)-LPETG-His6 (F43→TAG) and
pEVOL-BipRS(D286R) were co-transformed into BL21(DE3) for BipAla-incorporated
CD4 (D1-D2) expression. BipAla was added into 1 L LB culture to a final concentration
of 1 mM. The expression of BipRS was induced by 2 mM arabinose when OD600 reached
0.5, and the expression of CD4 was induced by 1 mM IPTG. BipAla-incorporated CD4
(D1-D2) was over-expressed in BL21 (DE3) and inclusion bodies were formed. A problem
involved in the expression was that BipAla was highly hydrophobic and there were a lot of
31
BipAla precipitates in the LB culture. The inclusion bodies were denatured and refolded
using the same protocol as that of wild-type CD4 (D1-D2).
The refolded protein was purified by the Ni-NTA affinity chromatography. The
chromatogram showed that the protein was eluted out as the concentration of imidazole
increased (Figure 11a). The SDS-PAGE result showed that full length BipAla-incorporated
CD4 (D1-D2) was successfully expressed and the majority of the refolded protein had one
disulfide bond (Figure 11b). Additionally, the UV260 value of this protein was significantly
higher than that of wild-type CD4 (D1-D2), possibly contributed by the increased
absorption of phenyl group at 260 nm. Afterwards, the Superdex 200 Increase 10/300 GL
SEC column was used to purify the protein, and we finally got 0.4 mg refolded protein
from 1 L LB.
32
Figure 11. Purification of BipAla-incorporated CD4 (D1-D2) by Ni-NTA affinity
chromatography. a, chromatogram of BipAla incoporated CD4 purified by Ni-NTA
column; b, SDS-PAGE gel analysis of protein sample before and after Ni-NTA purification.
B.C., before column; F.T., flow through.
a
b
33
3.2.2 IodoF incorporated CD4 (D1-D2)
The expression vectors pET22b-CD4(1-177)-LPETG-His6 (F43→TAG) and
pEVOL-IodoFRS were co-transformed into BL21 (DE3) for the expression of IodoF-
incorporated CD4 (D1-D2). The expression and refolding method was the same as that of
BipAla-incorporated CD4 (D1-D2). The water solubility of IodoF was much better than
BipAla and we got more inclusion bodies this time (25 mg inclusion bodies from 1 L LB).
A negative control group containing BL21 (DE3) co-transformed with the same plasmids
was also induced by IPTG and arabinose, but there was no IodoF added in the media. The
supernatant and inclusion bodies were separated after disruption of the bacteria and were
analyzed by SDS-PAGE (Figure 12). The full length protein was expressed in the inclusion
bodies only when there was Iodo-F in the environment. As the truncated protein CD4(1-
42) was only 4.7 kDa, it was not clearly visualized on the gel.
The refolded protein was purified by the Ni-NTA affinity chromatography. According
to the chromatogram and SDS-PAGE results (Figure 13), the full length protein was eluted
out in the fractions (9, 13). There were some dimers and impurity remaining in the protein,
so further purification by gel filtration was essential. The majority of the refolded IodoF-
incorporated protein was eluted out at around 17 mL, which was similar to wild-type CD4
(D1-D2). The left-shoulder of the peak indicated that the dimers was eluted out earlier
(Figure 13a). The SDS-PAGE gel showed that the fractions (36, 40) contained pure
monomer CD4(1-177)-LPETG-His6 (F43→IodoF) (Figure 14b). We collected these
fractions and measured the concentration. We finally got 1.6 mg refolded protein from 15
mg inclusion bodies. The yield of IodoF-incorporated CD4 (D1-D2) expression and
purification was around 2.7 mg refolded protein from 1 L LB culture.
34
Figure 12. Expression of IodoF-incorporated CD4 (D1-D2). (-), Negative control group
without IodoF addition; (+), Experimental group with IodoF. M, lane of protein marker; T,
total protein components; S, supernatant proteins; IB, inclusion body proteins.
35
Figure 13. Purification of IodoF-incorporated CD4 (D1-D2) by Ni-NTA affinity
chromatography. (a) Chromatogram of IodoF-incoporated CD4 (D1-D2) purified by Ni-
NTA column. (b) SDS-PAGE gel analysis of protein sample after Ni-NTA purification.
F.T., flow through.
a
b
36
Figure 14. Purification of IodoF-incorporated CD4 (D1-D2) by gel filtration. (a)
Chromatogram of IodoF-incoporated CD4 (D1-D2) purified by Superdex 200 10/300 GL
SEC column. (b) SDS-PAGE gel analysis of protein sample after gel filtration.
b
a
37
3.3 Summary
In summary, we have successfully expressed the wild-type CD4 (D1-D2) as inclusion
bodies in E.coli, and established a protocol for its in vitro refolding and purification. The
final yield was 4 mg/L LB. We then used the unnatural amino acid incorporation technique
to mutate Phe43 of CD4 to BipAla and IodoF, respectively. The yield of IodoF-
incorporated CD4 was excellent, about 2.7 mg/L LB. The yield of BipAla-incorporated
CD4 was only 0.4 mg/L LB, likely due to the poor solubility of BipAla. A potential
alternative is to incorporate bipyridylalanine, which is more hydrophilic than BipAla.
38
4. Sortase-Catalyzed Conjugation of CD4 (D1-D2) and IgG1-mim2
To produce enhanced eCD4-Ig by unnatural amino acid mutagenesis, our strategy was
to ligate the unnatural amino acid incorporated CD4 (D1-D2) produced above to the IgG1-
mim2 fragment by sortase-catalyzed protein-protein conjugation. Sortase A is a
transpeptidase that efficiently catalyzes the fusion of two proteins containing the
“LPXTG” sequence at the C-terminus of the first protein and the multi-glycine motif at the
N-terminus of the second protein. As described above, we have already generated CD4
(D1-D2) with the “LPXTG” sequence at the C-terminus. As a proof of concept, we first
examined the conjugation between CD4 (D1-D2) and the GFP protein containing an N-
terminal multiglycine motif, for GFP can be conveniently produced in E. coli and its
characteristic absorption at 488 nm can be used to facilitate verification of the fusion
product. If the conjugation is successful, we would continue to express IgG1-mim2 in 293T
cells and ligate it to CD4 (D1-D2).
4.1 Preliminary study of CD4 (D1-D2) –sfGFP assembly
4.1.1 Expression and purification of GB1-SortA-His6
The sortase A gene was incorporated into expression vector pET22b-GB1-SortA-His6,
which contains an immunoglobulin binding domain 1 (GB1) at the N-terminus to enhance
its solubility (Esposito and Chatterjee, 2006). This plasmid was transformed into BL21
(DE3) for sortase A expression. Sortase A was over-expressed in the form of soluble protein
and could be recovered from the supernatant after cell disruption.
The protein was purified Ni-NTA affinity chromatography. There was a strong peak
in the chromatogram and the SDS-PAGE gel showed that there are impurities at around 10
kDa (Figure 15). The fractions (6, 12) were collected for further purification by the Mono
Q 5/50 GL column. The isoelectric point of GB1-SortA-His6 was 6.82, so the protein was
negatively charged at pH 8.0 and could bind to the matrix of Mono Q column. The SDS-
39
PAGE result showed that the GB1-SortA-His6 was pure and migrated at around 27 kDa on
the tris-glycine gel (Figure 16).
40
Figure 15. Purification of GB1-SortA-His6 by Ni-NTA affinity chromatography. (a)
Chromatogram of GB1-SortA-His6 purified by Ni-NTA column. (b) SDS-PAGE gel
analysis of protein sample after Ni-NTA purification.
a
b
41
Figure 16. Purification of GB1-SortA-His6 by ion-exchange chromatography. (a)
Chromatogram of GB1-SortA-His6 purified by Mono Q column. (b) SDS-PAGE gel
analysis of protein sample after Mono Q purification.
a
b
42
4.1.2 Expression and purification of G5-sfGFP
Superfolder GFP (sfGFP) is a robustly folded variant of the green fluorescent protein.
The expression vector pET22b-G5-sfGFP-His6 was transformed into BL21 (DE3) for GFP
expression. A “GGGGG” motif was added at the N-terminus for the recognition by sortase
A. The molecular weight of G5-sfGFP-His6 is 27.9 kDa. The protein expression was
induced by IPTG when OD600 reached 0.6. Then the bacteria culture was incubated at 18° C
overnight. GFP was over-expressed in the form of soluble protein and could be recovered
from the supernatant after cell disruption.
The protein was purified by Ni-NTA affinity chromatography. Two UV280 peaks were
observed in the chromatogram, and there was also a strong UV488 peak. This additional
absorption peak at 488nm further indicated the successful expression of sfGFP, which is
excited at that wavelength. Based on these results, the fractions (9, 13) were collected
(Figure 17a). Afterwards, the protein was further purified by the HiTrap Q FF column. The
isoelectric point of sfGFP was 6.20, so the protein was negatively charged at pH 8.0 and
could bind to the Q column. The protein was eluted out as the concentration of sodium
chloride increased in the buffer (Figure 17b). The SDS-PAGE result showed that the 10
kDa impurity was removed from the protein by ion-exchange chromatography, but there
were still some upper bands, which may possibly represent GFP aggregates (Figure 17c).
After purification by gel filtration chromatography, the G5-sfGFP-His6 protein was
generally pure (Figure 18). The final yield of GFP expression and purification was around
2 mg from 1 L LB.
43
Figure 17. Purification of G5-sfGFP by Ni-NTA affinity chromatography and ion-
exchange chromatography. (a) Chromatogram of G5-sfGFP purified by Ni-NTA column.
(b) Chromatogram of G5-sfGFP purified by Q column. (c) SDS-PAGE gel analysis of
protein sample after Q column purification. B.C., before column.
b
a
c
44
Figure 18. Purification of G5-sfGFP by gel filtration. (a) Chromatogram of G5-sfGFP
purified by Superdex 200 10/300 GL SEC column. (b) SDS-PAGE gel analysis of protein
sample after gel filtration.
b
a
45
4.1.3 CD4 (D1-D2)-sfGFP assembly under different conditions
Next, we attempted to identify an optimal condition for sortase-catalyzed conjugation o
CD4 (D1-D2) and sfGFP (Figure 19a). We first examined whether the sortase A
concentration could affect the efficiency of conjugation. To optimize the sortase A-
catalyzed conjugation reaction, we first set up small-scale (5 μl) reactions. The
concentrations of sortase A utilized in the reaction were 0, 15, 30 and 60 µ M, while 100
μM CD4(1-177)-LPETG-His6 and 40 μM G5-sf-GFP were used in the reaction. All these
components were present in a reaction buffer which contained 10mM CaCl ₂, 150mM NaCl,
and 50mM Tris (pH 8.0). The reaction buffer additionally contained calcium, which is an
essenetial element for catalysis by SortA. The reactants and enzyme were incubated at
37 ° C for 2 hours. Afterwards, the samples were treated at 95 ° C for 10 minutes and
analyzed by SDS-PAGE (Figure 19b). The result showed that the yield of the fusion
product increased when there was higher concentration of sortase A. The band of the fusion
product was at nearly 50 kDa, which corresponded with its theoretical molecular weight,
47.9 kDa. Therefore, we decided to use 60 μM sortase A for the fusion protein assembly.
46
Figure 19. CD4 (D1-D2)-sfGFP protein assembly catalyzed by sortase A. (a) Diagram
of the CD4 (D1-D2)-sfGFP fusion reaction. (b) SDS-PAGE analysis of CD4 (D1-D2)-
sfGFP fusion reactions under different SortA concentrations.
b
a
47
4.1.4 Verification of CD4 (D1-D2)-sfGFP fusion product
We scaled up the reaction to 100 μL (100 μM CD4 (D1-D2), 40 μM G5-sfGFP and 60
μM GB1-SortA-His6). The excess reactants, enzyme and fusion product could be separated
by ion-exchange chromatography because of their different isoelectric points. Therefore,
we used the Mono Q 5/50 GL column to separate and verify the proteins (Figure 20a).
The binding buffer utilized for Mono Q column was 25mM Tris (pH 8.0) and the
elution buffer was 1 M NaCl, 25 mM Tris (pH 8.0). The pI values of CD4 (D1-D2), G5-
sfGFP, GB1-SortA-His6 and the fusion product were 8.84, 6.20, 6.82 and 6.66 respectively.
At pH 8.0, CD4 was positively charged and did not bind Mono Q column. The other
proteins were negatively charged and could bind to the column matrix well. The gradient
was set as 0-30% elution buffer, 20 mL. Excess CD4 (D1-D2) was eluted out first, followed
by GB1-SortA-His6, CD4 (D1-D2) -sfGFP conjugate and excess G5-sfGFP. All the
proteins were well separated for distinct peaks were observed for each protein. The UV488
absorption also indicated that the third and fourth peaks are GFP-related protein (Figure
20a). Based on the calculation of the AUC of UV280, the yield of the conjugation reaction
was around 46% regarding GFP. The fractions at the peaks were collected for SDS-PAGE
analysis (Figure 20b). The result corresponded with our assumption that the proteins were
eluted out in a sequence of CD4 (D1-D2), GB1-SortA-His6, conjugation product and G5-
sfGFP separately. Thus, the fusion product and excess reactants could be well recovered
by ion-exchange chromatography.
48
Figure 20. Verification and separation of CD4 (D1-D2)-sfGFP assembly product by
ion-exchange chromatography. (a) Chromatogram of the excess reactants and reaction
products separated by Mono Q column. (b) SDS-PAGE analysis of the protein peaks eluted
from Mono Q column.
b
a
49
4.2 CD4 (D1-D2) –IgG1-mim2 assembly catalyzed by sortase A
4.2.1 Expression and purification of G5-IgG1-mim2
The C-terminus tyrosine sulfation on the CCR5 has been characterized and proven to
facilitate HIV entry (Farzan et al., 1999). In the eCD4-IgG design, the CCR5 mimetic
peptides were tyrosine sulfated (Farzan et al., 2000; Gardner et al., 2014). Therefore, the
Tyrosylprotein Sulfotransferase 2 (TPST2) was introduced into the expression process of
G5-IgG1-mim2 fragment to induce the posttranslational modification on the CCR5
mimetic peptide.
The expression vectors CMVR-G5-IgG1-mim2 (64 μg/dish) and pcDNA(3.1+)–
TPST2 (16 μg/dish) were co-transfected 293T cells in three 30mL dishes containing 293T
cells. These embryonic kidney cell lines were cultured in 293 freestyle media to facilitate
protein expression. Following transfection, the 293T cells would express the protein and
secrete the final refolded and soluble product into the supernatant. The cell supernatant was
collected twice following the fourth and eighth days of incubation.
After the supernatant collection, the media was concentrated and the expression
products were analyzed by SDS-PAGE (Figure 21). There were still large quantity of
proteins expressed in the second round of protein expression (Day 5-8). Afterwards, the
G5-IgG1-mim2 was purified by affinity chromatography using the immunoglobulin
specific Protein A column. The protein was purified using 1× PBS as the binding buffer and
acidic solution as elution buffer (0.1M citric acid, 150mM NaCl, pH=3.0). From the
chromatogram we could see a strong protein peak, and the gel analysis showed that the
protein migrated slightly over 50kD, which corresponded with the theoretical G5-IgG1-
mim2 protein size of 56.9 kD (Figure 21). The Protein A fractions (6, 10) were collected
for further purification by gel filtration. The SDS-PAGE results indicated that the G5-IgG1-
mim2 protein was generally pure (Figure 22), and the total yield was 2.7 mg from 180 mL
293 freestyle media.
50
Figure 21. Expression of G5-IgG1-mim2 and protein purification by Protein A affinity
chromatography. (a) Chromatogram of G5-IgG1-mim2 purified by Protein A column. (b)
SDS-PAGE gel analysis of protein sample before and after Protein A purification. Lanes
(1, 2) shows the sample from the two rounds of expression separately. F.T., flow through.
b
a
51
Figure 22. Purification of G5-IgG1-mim2 by gel filtration. (a) Chromatogram of G5-
IgG1-mim2 purified by Superdex 200 10/300 GL column. (b) SDS-PAGE gel analysis of
protein sample after Protein A purification.
a
b
52
4.2.2 Expression and purification of SortA
The GB1-SortA-His6 was used in the preliminary study of CD4 (D1-D2)-sfGFP.
However, the GB1 domain could interact with the IgG1 Fc fragment and affect the sortase
A catalysis. Therefore, we constructed the pET28b-His6-SUMO-SortA for sortase A
expression. The small ubiquitin-like modifier (SUMO) propeptide was added to enhance
the protein expression and a His6 tag was added at the N-terminus of the protein for
purification. The expression vector was transformed into BL21 (DE3), and the protein
expression was induced by IPTG when OD600 reached 0.6. Then the bacteria culture was
incubated at 18° C overnight. The His6-SUMO-SortA was over-expressed in the form of
soluble protein and could be recovered from the supernatant after cell disruption.
The His6-SUMO-SortA protein was purified Ni-NTA affinity chromatography. There
was a strong peak in the chromatogram and the SDS-PAGE gel showed that the His6-
SUMO-SortA migrated at around 35 kD while there are impurities at around 10 and 75
kDa (Figure 23). The fractions (5, 11) were collected and cleaved by ulp1 protease into
His6-SUMO and SortA. The protein was purified by Ni-NTA affinity chromatography
again and the sortase A without His6 tag was observed in the flow through solution (Figure
24b). Sortase A was further purified by gel filtration (Figure 24a). The SDS-PAGE result
showed that the SortA protein was pure and migrated at around 17 kDa on the tris-glycine
gel (Figure 24b). The yield of the SortA expression and purification was around 53 mg
from 1 L LB.
53
Figure 23. Purification of His6-SUMO-SortA by Ni-NTA chromatography. (a)
Chromatogram of His6-SUMO-SortA purified by Ni-NTA column. (b) SDS-PAGE gel
analysis of protein sample after Ni-NTA purification. B.C., before column. F.T., flow
through.
a
b
54
Figure 24. Purification of SortA by Ni-NTA chromatography and gel filtration. (a)
Chromatogram of SortA purified by Superdex 200 16/600 GL SEC column. (b) SDS-
PAGE gel analysis of protein sample after purification. The protein sample after ulp1
cleavage was purified by Ni-NTA column (Lane 1-2), and the flow through containing
SortA was further purified by gel filtration (Lane 3-9).
a
b
55
4.2.3 Fusion reaction of CD4 (D1-D2) and G5-IgG1-mim2
Based on the preliminary study of CD4 (D1-D2)-sfGFP assembly, a satisfying yield
can be achieved using 100 μM CD4 (D1-D2) and 40 μM G5-sfGFP, catalyzed by 60 μM
SortA. As the IgG1 Fc fragment is a dimerized protein, two CD4 (D1-D2) molecules could
be conjugated onto one IgG1-mim2 molecule. Therefore, 100 μM CD4(1-177)-LPETG-
His6 and 20 μM G5-IgG1-mim2 were utilized in the protein fusion reaction. Sortase A
concentrations ranging from 15 μM to 90 μM were tested. The reaction temperature was
37 ° C. The protein ligation results were analyzed by SDS-PAGE (Figure 25). For all the
reaction groups, there was a clear band slightly higher than 75 kD and another band above
100 kD. The theoretical protein size of the primary and secondary conjugation products are
77 and 97 kD, respectively. The gel analysis indicated that we were able to fuse two CD4
(D1-D2) molecules with one G5-IgG1-mim2 fragment, and more secondary conjugation
products were synthesized with a higher sortase A concentration. In addition to optimizing
the Sort A concentration, we also attempted to modify the reaction time to enhance the
formation of the CD4 ₂-IgG fusion product. Fusion reactions catalyzed by 60 μM SortA
were performed for 6 or 24 hours, and the gel result indicated that the yield of this reaction
were similar to the 2 hour reaction. These fusion reaction results demonstrated that CD4
(D1-D2)–IgG1-mim2 assembly catalyzed by sortase A was feasible, but optimization of
reaction conditions is still required to achieve higher yield of the secondary conjugation
product.
56
Figure 25. SDS-PAGE analysis of CD4 (D1-D2)-IgG1-mim2 fusion reaction catalyzed
by sortase A.
57
4.3 Summary
In this section, we focused on using sortase A to conjugate CD4 and IgG-mim2. We
first utilized the fluorescent protein sfGFP as a substitute for IgG-mim2 in order to optimize
the sortase-catalyzed protein fusion. We found that CD4 (D1-D2) was successfully
conjugated to G5-sfGFP-His6 under the catalysis of sortase A at a yield of nearly 50%.
Based on this finding, we continued to conjugate CD4 to IgG1-mim2. To this end, we used
293T cells to express IgG1-mim2 with an N-terminal pentaglycine tag, referred to as G5-
IgG1-mim2. After purification by Protein A affinity chromatography and gel filtration, we
were able to obtain 2.7 mg G5-IgG1-mim2 from 180 mL 293 freestyle media. Finally, we
showed that CD4 (D1-D2) and G5-IgG1-mim2 were conjugated under the catalysis of
sortase A. We will continue to optimize this reaction, for example, by using a higher CD4
(D1-D2):IgG1-mim2 ratio and/or a higher concentration of sortase A.
58
5. Conclusion
This project aimed to enhance the anti-HIV efficacy of eCD4-Ig through unnatural
amino acid mutagenesis at the Phe43 site. To accomplish our objective, we used a three-
step approach. The first step was to mutate Phe43 of CD4 (D1-D2) to the unnatural amino
acid BipAla or IodoF by using an aminoacyl-tRNA synthetase and amber-suppressor-tRNA
pair specific for incorporating these unnatural amino acids at a site-specific location of a
protein. We were able to express the protein as inclusion bodies in E.coli and then refold it
in vitro. The yield of IodoF-incorporated CD4 (D1-D2) was very good (2.7 mg from 1L
LB), only slightly less than that that of wild-type CD4 (D1-D2) protein (4 mg from 1L LB).
However, the yield of BipAla-incorporated CD4 (D1-D2) was low (0.4 mg from 1L LB),
likely due to the poor solubility of BipAla. The second step was to express the G5-IgG1 Fc
fragment fused with CCR5mim2 sulfopeptide in 293T cells. After purification, we were
able to obtain the protein at 2.7 mg from 180 mL 293 freestyle media. In the third step, we
used sortase to conjugate CD4 (D1-D2) and IgG1-mim2.
Despite these progresses, there are still several issues that need to be addressed in
future studies. First, the extremely low solubility of BipAla made it difficult to be
efficiently incorporated. In the future, we plan to incorporate bipyridylalanine, which is as
large as BipAla but much more water soluble. Second, although CD4 (D1-D2)-IgG1-mim2
fusion was successfully produced by sortase A catalysis, the yield was still low. We plan to
optimize the reaction by either using sortase A at a higher concentration, increasing the
ratio of CD4 to IgG1-mim2, and/or extending the reaction time.
If these eCD4-Ig variants are successfully produced on a large scale, we will then
measure their binding affinity for gp120 by surface plasmon resonance (SPR) analysis. We
expect that our research will lead to the development of enhanced eCD4-Ig variants with
stronger binding affinity for gp120.
59
6. References
Barouch, D. H. (2008). Challenges in the development of an HIV-1
vaccine. Nature 455(7213), 613.
Cerutti, N., Killick, M., Jugnarain, V ., Papathanasopoulos, M., and Capovilla, A. (2014).
Disulfide reduction in CD4 domain 1 or 2 is essential for interaction with HIV
glycoprotein 120 (gp120), which impairs thioredoxin-driven CD4 dimerization. Journal
of Biological Chemistry 289(15), 10455-10465.
Choe, H., Farzan, M., Sun, Y ., Sullivan, N., Rollins, B., Ponath, P. D., Wu, L., Mackay,
C.R., LaRosa, G., Newman, W. and Gerard, N. (1996). The β-chemokine receptors CCR3
and CCR5 facilitate infection by primary HIV-1 isolates. Cell 85(7), 1135-1148.
Cocchi, F., DeVico, A. L., Garzino-Demo, A., and Arya, S. K. (1995). Identification of
RANTES, MIP-1alpha, and MIP-1beta as the major HIV-suppressive factors produced by
CD8positive T cells. Science 270(5243), 1811.
Daar, E. S., Li, X. L., Moudgil, T., and Ho, D. D. (1990). High concentrations of
recombinant soluble CD4 are required to neutralize primary human immunodeficiency
virus type 1 isolates. Proceedings of the National Academy of Sciences 87(17), 6574-
6578.
Dalgleish, A. G., Beverley, P. C., Clapham, P. R., Crawford, D. H., Greaves, M. F., and
Weiss, R. A. (1984). The CD4 (T4) antigen is an essential component of the receptor for
the AIDS retrovirus. Nature 312(5996), 763-767.
Esposito, D., and Chatterjee, D. K. (2006). Enhancement of soluble protein expression
through the use of fusion tags. Current opinion in biotechnology, 17(4), 353-358.
Feng, Y ., Broder, C. C., Kennedy, P. E., and Berger, E. A. (1996). HIV-1 Entry Cofactor-
Functional cDNA cloning of a seven-transmembrane, G protein-coupled
60
receptor. Science, 272(5263), 872-877.
Fletcher, C. V ., DeVille, J. G., Samson, P. M., Moye Jr, J. H., Church, J. A., Spiegel, H.
M., Palumbo, P., Fenton, T., Smith, M.E., Graham, B. and Kraimer, J. M. (2007).
Nonlinear pharmacokinetics of high-dose recombinant fusion protein CD4-IgG2 (PRO
542) observed in HIV-1–infected children. The Journal of allergy and clinical
immunology, 119(3), 747.
Friedman-Kien, A. E., Laubenstein, L., Marmor, M., Hymes, K., Green, J., Ragaz, A.,
Gottleib, J., Muggia, F., Demopoulos, R. and Weintraub, M. (1981). Kaposis sarcoma and
Pneumocystis pneumonia among homosexual men--New York City and
California. MMWR, 30(25), 305-8.
Gallo, R. C., Salahuddin, S. Z., Popovic, M., Shearer, G. M., Kaplan, M., Haynes, B. F.,
Palker, T.J., Redfield, R., Oleske, J., Safai, B. and White, G. (1982). Frequent detection
and isolation of cytopathic retroviruses (HTLV-III). N. Engl. J. Med, 306, 248.
Gardner, M. R., Kattenhorn, L. M., Kondur, H. R., V on Schaewen, M., Dorfman, T.,
Chiang, J. J., Haworth, K.G., Decker, J.M., Alpert, M.D., Bailey, C.C. and Neale Jr, E. S.
(2015). AA V-expressed eCD4-Ig provides durable protection from multiple SHIV
challenges. Nature, 519(7541), 87.
Gaschen, B., Taylor, J., Yusim, K., Foley, B., Gao, F., Lang, D., Novitsky, V ., Haynes, B.,
Hahn, B.H., Bhattacharya, T. and Korber, B. (2002). Diversity considerations in HIV-1
vaccine selection. Science, 296(5577), 2354-2360.
Hussey, R. E., Richardson, N. E., Kowalski, M., Brown, N. R., Chang, H. C., Siliciano,
R. F., Dorfman, T., Walker, B., Sodroski, J. and Reinherz, E. L. (1988). A soluble CD4
protein selectively inhibits HIV replication and syncytium formation. Nature, 331(6151),
78-81.
Hober, S., Nord, K., and Linhult, M. (2007). Protein A chromatography for antibody
61
purification. Journal of Chromatography B, 848(1), 40-47.
Kim, C. H., Axup, J. Y ., and Schultz, P. G. (2013). Protein conjugation with genetically
encoded unnatural amino acids. Current opinion in chemical biology, 17(3), 412-419.
Klatzmann, D., Barre-Sinoussi, F., Nugeyre, M. T., Dauguet, C., Vilmer, E., Griscelli, C.,
Brun-Vezinet, F., Rouzioux, C., Gluckman, J.C., Chermann, J.C. and Montagnier, L.
(1984). Selective tropism of lymphadenopathy associated virus (LAV) for helper-inducer
T lymphocytes. Science, 225, 59-64.
Klatzmann, D., Champagne, E., Chamaret, S., Gruest, J., Guetard, D., Hercend, T.,
Gluckman, J.C. and Montagnier, L. (1984). T-lymphocyte T4 molecule behaves as the
receptor for human retrovirus LAV . Nature, 312(5996), 767-768.
Kwong, P. D., Wyatt, R., Robinson, J., and Sweet, R. W. (1998). Structure of an HIV
gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human
antibody. Nature, 393(6686), 648.
Laskey, S. B. and Siliciano, R. F. (2014). A mechanistic theory to explain the efficacy of
antiretroviral therapy. Nature reviews. Microbiology, 12(11), 772.
Levary, D. A., Parthasarathy, R., Boder, E. T., and Ackerman, M. E. (2011). Protein-
protein fusion catalyzed by sortase A. PloS one, 6(4), e18342.
Mao, H., Hart, S. A., Schink, A., and Pollok, B. A. (2004). Sortase-mediated protein
ligation: a new method for protein engineering. Journal of the American Chemical
Society, 126(9), 2670-2671.
Mazmanian, S. K., Liu, G., Ton-That, H., and Schneewind, O. (1999). Staphylococcus
aureus sortase, an enzyme that anchors surface proteins to the cell
wall. Science, 285(5428), 760-763.
Rizzuto, C. D., Wyatt, R., Herná ndez-Ramos, N., Sun, Y ., Kwong, P. D., Hendrickson, W.
62
A., and Sodroski, J. (1998). A conserved HIV gp120 glycoprotein structure involved in
chemokine receptor binding. Science, 280(5371), 1949-1953.
Starcich, B. R., Hahn, B. H., Shaw, G. M., McNeely, P. D., Modrow, S., Wolf, H., Parks,
E.S., Parks, W.P., Josephs, S.F., Gallo, R.C. and Wong-Staal, F. (1986). Identification and
characterization of conserved and variable regions in the envelope gene of HTLV-
III/LA V , the retrovirus of AIDS. Cell, 45(5), 637-648.
Stricher, F., Huang, C. C., Descours, A., Duquesnoy, S., Combes, O., Decker, J. M., Do
Kwon, Y ., Lusso, P., Shaw, G.M., Vita, C. and Kwong, P. D. (2008). Combinatorial
optimization of a CD4-mimetic miniprotein and cocrystal structures with HIV-1 gp120
envelope glycoprotein. Journal of molecular biology, 382(2), 510-524.
Tintori, C., Selvaraj, M., Badia, R., Clotet, B., Esté , J. A., and Botta, M. (2013).
Computational Studies Identifying Entry Inhibitor Scaffolds Targeting the Phe 43 Cavity
of HIV ‐1 gp120. ChemMedChem, 8(3), 475-483.
Wang, L., Magliery, T. J., Liu, D. R., & Schultz, P. G. (2000). A New Functional
Suppressor tRNA/Aminoacyl− tRNA Synthetase Pair for the in Vivo Incorporation of
Unnatural Amino Acids into Proteins. Journal of the American Chemical
Society, 122(20), 5010-5011.
Wilen, C. B., Tilton, J. C., and Doms, R. W. (2012). Molecular mechanisms of HIV entry.
In Viral Molecular Machines (Springer US), pp. 223-242.
Wyatt, R., Kwong, P. D., Desjardins, E., and Sweet, R. W. (1998). The antigenic structure
of the HIV gp120 envelope glycoprotein. Nature, 393(6686), 705.
Xie, J., & Schultz, P. G. (2006). A chemical toolkit for proteins—an expanded genetic
code. Nature Reviews Molecular Cell Biology, 7(10).
Young, T. S., Ahmad, I., Yin, J. A., and Schultz, P. G. (2010). An enhanced system for
unnatural amino acid mutagenesis in E. coli. Journal of molecular biology, 395(2), 361-
63
374.
Yu, X., Talukder, P., Bhattacharya, C., Fahmi, N. E., Lines, J. A., Dedkova, L. M.,
LaBaer, J., Hecht, S.M. and Chen, S. (2014). Probing of CD4 binding pocket of HIV-1
gp120 glycoprotein using unnatural phenylalanine analogues. Bioorganic & medicinal
chemistry letters, 24(24), 5699-5703.
Zhang, W., Canziani, G., Plugariu, C., Wyatt, R., Sodroski, J., Sweet, R., Kwong, P.,
Hendrickson, W. and Chaiken, I. (1999). Conformational changes of gp120 in epitopes
near the CCR5 binding site are induced by CD4 and a CD4 miniprotein
mimetic. Biochemistry, 38(29), 9405-9416.
Zhou, T., Xu, L., Dey, B., Hessell, A. J., Van Ryk, D., Xiang, S. H., Yang, X., Zhang,
M.Y ., Zwick, M.B., Arthos, J. and Burton, D. R. (2007). Structural definition of a
conserved neutralization epitope on HIV-1 gp120. Nature, 445(7129), 732.
Abstract (if available)
Abstract
Human immunodeficiency virus (HIV) entry into the host cell is initiated by the sequential binding of the viral envelope protein gp120 to the host cell’s receptor CD4 and co-receptor CCR5 or CXCR4. Recently Dr. Farzan’s group developed a novel HIV entry inhibitor eCD4-Ig by fusing a CD4 fragment and a small CCR5-mimetic sulfopeptide to the N- and C-termini of an IgG Fc fragment. It was shown that eCD4-Ig was able to bind avidly and cooperatively to gp120 and that its anti-HIV breadth and potency were better than the currently characterized broadly neutralizing antibodies to HIV-1. The research goal of our project was to further enhance the anti-HIV potency of eCD4-Ig by unnatural amino acid mutagenesis. Based on the crystal structure of CD4-gp120 complex, Phe43 of CD4 is surrounded by a large hydrophobic cavity of gp120. Therefore, we propose to mutate Phe43 to a larger unnatural amino acid such as biphenylalnine (BipAla) and iodo-phenylalanine (IodoF), which can potentially enhance the binding between eCD4-Ig and gp120. To achieve this objective, we combined two novel protein chemistry techniques: one is the site-specific incorporation of unnatural amino acids into recombinant proteins, the other is the sortase-catalyzed protein-protein conjugation. So far I have successfully expressed CD4 (D1-D2) variants containing BipAla or IodoF in E. coli, and expressed the IgG1 Fc fragment fused with CCR5mim2 sulfopeptide mim2 (IgG1-mim2) in mammalian cells. I have also successfully used sortase to conjugate CD4 (D1-D2) and IgG1-mim2, although the yield is still low. In future work, it is necessary to further optimize the conjugation between CD4 (D1-D2) and IgG1-mim2, and measure the binding affinity of the resulting eCD4-Ig variants for gp120.
Linked assets
University of Southern California Dissertations and Theses
Conceptually similar
PDF
Site-specific PEGylation of recombinant immunotoxin αFAP-PE38 for half-life extension
PDF
Site-specific PEGylation of recombinant immunotoxin antiFAP-CD8h-PE38 with an expanded genetic code
PDF
Enhancing the specificity and cytotoxicity of chimeric antigen receptor Natural Killer cells
PDF
Antibodies and elastin-like polypeptides: cellular and biophysical characterization of an anti-ELP monoclonal and an anti-CD3 single-chain-ELP fusion
PDF
pH-sensitive cytotoxicity of a cell penetrating peptide fused with a histidine-glutamate co-oligopeptide
PDF
Cell penetrating peptide-based drug delivery system for targeting mildly acidic pH
PDF
Factors that may impact drug disposition and metabolism
PDF
Y-shaped DNA based pMHC nonamers for detecting low-affinity T cells
PDF
Towards DNA-directed assembly of pMHC multimers for detection of low-affinity T cells
PDF
Development of new approaches for antibody modification
PDF
Deregulation of CD36 expression in cancer presents a potential targeting therapeutic opportunity
PDF
Characterization of the interaction of nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein 12 (Nlrp12) with hematopoietic cell kinase (Hck)
PDF
Short term high fat diet (HFD) stimulates β cell proliferation through mTOR while the prolonged treatment induces β cell senescence via p27
PDF
Developing recombinant single chain Fc-dimer fusion proteins for improved protein drug delivery
PDF
Mining the felinone A biosynthetic pathway
PDF
Characterization of the transferrin oligomer and its potential application in drug delivery
PDF
Pseudotyped viral vectors: HIV gene therapy applications and basic studies of SARS-COV-2
PDF
Protein ADP-ribosylation: from biochemical characterization to therapeutic applications
PDF
Role of purinergic P2X7 receptors in inflammatory responses in the brain and liver: a study using a mouse model of chronic ethanol and high-fat diet exposure
Asset Metadata
Creator
Chen, Manyun
(author)
Core Title
Enhancing the anti-HIV potency of eCD4-Ig by unnatural amino acid mutagenesis
School
School of Pharmacy
Degree
Master of Science
Degree Program
Pharmaceutical Sciences
Publication Date
08/02/2017
Defense Date
06/22/2017
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
CD4,HIV gp120,OAI-PMH Harvest,sortase A,unnatural amino acid
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Xie, Jianming (
committee chair
), Okamoto, Curtis (
committee member
), Shen, Wei-Chiang (
committee member
)
Creator Email
manyunch@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c40-421975
Unique identifier
UC11264161
Identifier
etd-ChenManyun-5673.pdf (filename),usctheses-c40-421975 (legacy record id)
Legacy Identifier
etd-ChenManyun-5673.pdf
Dmrecord
421975
Document Type
Thesis
Rights
Chen, Manyun
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
CD4
HIV gp120
sortase A
unnatural amino acid