1. 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) 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 88 th 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. 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 domain- derived 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. 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. 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. 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. Sequencing continues to be uninformative at this time. Also antibody binding affinity studies have not yet been conducted. Conclusion 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 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. 1 1kb L Lane 1: gel purified N88.1 overlap PCR product. Lane 1: gel purified N88.2 overlap PCR product. Overlap PCR created products of The correct molecular weight Results 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 3 4 1kb L Lane 1: EcoR1 digested N88 Lane 2: SfiI digested N88 after Gel purification 1 2 1kb L The dgODg plasmids were successfully linearized. EcoR1 indicated single digest, Sfi1 double digested vectors. Fig. 1: Gp120 structure, “Structure of the Core of the HIV-1 gp120 Exterior Envelope Glycoprotein”. Fig. 2: Diagram of Overlap Extension PCR, “Nature Methods”. gp120 Alexa Fluor 488 PE antibody Fig. 3: Surface Display for FACS. From Piyali Saha.