Transcript Library Creation and TA Cloning for HIV gp120

Library Creation and TA Cloning for HIV gp120 N88 Mutants
Kris Barnes (SUNY Oswego), Piyali Saha (IISc), Raghavan Varadajaran(IISc)
Indian Institute of Science, Bangalore
Introduction
HIV gp120 is the primary protein responsible for binding to CD4
proteins on immune cells facilitating viral entry into the cell. It is roughly
120 kd in size and about 500 amino acids long. It is composed of an outer
domain which contains the CD4 binding site as well as an inner domain. It
is typically bound to gp41, a HIV transmembrane protein, to form trimers
which are part of the overall envelope spike necessary for viral entry into
cells. (Saha et al. 2012)
Fig. 1: Gp120 structure,
“Structure of the Core of the
HIV-1 gp120 Exterior Envelope
Glycoprotein”.
HIV gp120 contains numerous glycosylation sites, including four on
the inner domain and ~20 on the outer domain. Outer domain
glycosylation sites have been indicated as contributing to evasion of
neutralizing antibodies as well as overall reduced protein stability. When
they are removed antibody binding affinity has been shown to increase
significantly. (Bhattacharyya et al. 2010) However, similar studies on the
effects of inner domain glycosylation are lacking, and this may also prove
to have a significant effects on protein stability.
Fluorescence Assisted Cell Sorting is a method using flow cytometry
to analyze and sort individual cells based on specific properties.
Specifically in this study, it can be used to determine overall surface
expression of proteins as well as binding affinity to antibodies. It
measures fluorescence of individual cells, and can also sort them in order
to aid in the creation of a mutant library.
Objective
In this experiment we aim to create yeast expressing 88th position site
specific mutant gp120 protein (N88 mutants) in order to to elucidate the
effect of the removal of an important glycosylation site in the inner
domain on the overall stability of the protein. We will test the properties
of the mutant protein through FACS analysis.
Materials and Methods
Overlap PCR (OEP) was used to create site specific mutants at the
N88 position. Initially deep vent PCR was used to create two products,
one with a vector specific reverse primer and N88 forward primer, and
one with N88 reverse primers containing the mutation as well as vector
specific forward primer. OEP used vector specific primers to create one
final product containing the mutation.
Fig. 2: Diagram of Overlap Extension
PCR, “Nature Methods”.
1 1kb L
Lane 1: gel purified
N88.1 overlap PCR
product.
Results
1 1kb L
1 2 3 4 1kb L
SfiI successful
digest
Lane 1: Undigested
N88 pPNLS vector
Lane 2: EcoRI
digested N88 (7.5
kb)
Lane 3: SfiI
digested N88
pPNLS vector (6.4
kb)
Lane 4: same as
lane 3
1 2 1kb L
Lane 1: EcoR1 digested
N88
Lane 2: SfiI digested
N88 after
Gel purification
Lane 1: gel purified
N88.2 overlap PCR
product.
The dgODg plasmids were successfully
linearized. EcoR1 indicated single digest,
Overlap PCR created products of Sfi1 double digested vectors.
The correct molecular weight
For transformation a XLIblue strain e-coli were heat shocked in the
presence of N88 dgODg plasmids and allowed to recover. Qiagen Plasmid
Purification protocol was used to obtain purified plasmid.
Yeast strain Eby100 was used for yeast homologous recombination.
Transformation was done using Li Acetate method. A vector : insert ratio
of 1:4 was used, with 500 ng of Vector used total. Cells were plated in
SDCAA media (pH 4.5).
Protein stability was checked using Fluorescence Assisted Cell Sorting
(FACS). Samples were prepared by incubating different mutant cells with
Anti-MYC antibodies to test for gp120 surface expression. Respective
secondary antibody were used and sample fluorescence was tested with
FACS. Mutant cells were sorted according to fluorescence as well, in order
to help create a library for antibody binding studies in the future.
PE
antibody
Alexa Fluor
488
Fig. 3: Surface Display for FACS.
From Piyali Saha.
FACS results showed high levels of gp120
surface expression in mutant cells. This
held true at higher cell counts during cell
sorting. Shown here, clockwise from top
left, are a) uninduced N88 cells, b) wild
type N88 cells, and c) mutant N88 cells.
Graphs measure fluorescence levels
(protein expression) vs. cell count.
Sequencing continues to be uninformative at this time. Also antibody
binding affinity studies have not yet been conducted.
Conclusion
For TA cloning Fermentas insTAclone TA cloning kit was used. PCR of
overlap products with TAQ polymerase created poly-T ends and allowed
for ligation using T4 DNA ligase. Cells were plated on x-gal and cells
containing inserts were grown in 96 well LB/amp incubation plates, and
replated on to rectangular colony analysis plate. Sanger sequencing
method was used.
References
1) “Structure of the Core of the HIV-1 gp120 Exterior Envelope Glycoprotein”, http://www.hiv.lanl.gov/content/sequence/HIV/REVIEWS/Sodroski.html,
2) “Nature Methods”, http://www.nature.com/nprot/journal/v2/n4/fig_tab/nprot.2007.132_F1.html
3) Bhattacharyya, S., Rajan, R. E., Swarupa, Y., Rathore, U., Verma, A., Udaykumar, R., and Varadarajan, R. (2010) Design of a non-glycosylated outer domainderived HIV-1 gp120 immunogen that binds to CD4 and induces neutralizing antibodies. J Biol Chem 285, 27100-10
4) Saha, P., Bhattacharyya, S., Kesavardhana, S., Miranda, E. R., Ali, P. S., Sharma, D., and Varadarajan, R. (2012) Designed cyclic permutants of HIV-1 gp120:
implications for envelope trimer structure and immunogen design. Biochemistry 51, 1836-47.
All PCR products appear to have been created successfully, include
overlap products. Ligation of overlap products appears to have been
successful in both yeast homologous recombination and TA cloning.
However sequencing results from TA cloning are uninformative. FACS
results demonstrate a high level of gp120 surface expression for the
library cells relative to WT cells. No results related to antibody binding
affinity have been obtained yet.
Acknowledgements
-RV lab -IISc
-SUNY Oswego OIEP and
the Global Laboratory Initiative
-Shashi Kanbur