The expression, purification and characterisation of recombinant HIV-1 subtype C gp120

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dc.contributor.author Michler, Katherine Laura
dc.date.accessioned 2008-10-17T10:52:52Z
dc.date.available 2008-10-17T10:52:52Z
dc.date.issued 2008-10-17T10:52:52Z
dc.identifier.uri http://hdl.handle.net/10539/5763
dc.description.abstract HIV-1, the virus that causes AIDS, is spreading at an alarming rate. Subtype C, which accounts for approximately 50% of infections worldwide, and 98% of infections in Southern Africa, is by far the most prevalent form of the virus. Most molecular and biochemical studies have been performed on HIV-1 subtype B isolates and products, however, and there is a relative scarcity of corresponding data on subtype C. It is therefore of crucial importance to study subtype C HIV-1 strains in order to understand their characteristic pathogenic effects and to develop effective treatment strategies. The aim of research in our laboratory is the development of novel treatment strategies, with particular focus on identifying novel Subtype C Env-binding peptide ligands. This necessitates the development of reagents for use in the discovery and testing of these compounds. In line with this, the aim of this project was the production and characterisation of recombinant Subtype C gp120s generated from a recently compiled HIV-1 virus cohort. To this end, the gp160-coding regions of 20 South African Subtype C HIV-1 strains isolated from AIDS patients presenting at the Johannesburg General hospital in 2005 were amplified by PCR and sequenced. The gp160 amplicons were used to amplify and clone the gp120-encoding regions of these isolates. Two clones, pTriEx- FV3 and pTriEx-FV5, originating from CXCR4- and CCR5-utilising strains respectively, were selected for further use. These clones were cotransfected into insect cells together with a baculoviral DNA backbone in order to generate gp120-expressing baculoviruses by homologous recombination. Recombinant baculoviruses were used to infect Sf9 insect cell cultures for expression of recombinant gp120, which was then purified using a combination of lectin affinity chromatography and ion exchange chromatography. In order to determine the functionality and conformational integrity of the recombinant gp120, the ability of these purified gp120s to bind CD4 and a panel of well-characterised monoclonal antibodies against various epitopes on gp120 (F425 A1g8, 2G12, F425 B4a1, F425 B4e8, 48d, 17b, IgG1 b12, 5F7, 4G10, 9301, ID6, Chessie 13-39.1, 654-30D and 670-30D) was assessed. Gp120 from the CXCR4-using isolate, FV3, appeared to have an intact, functional CD4 binding site as measured by its ability to bind to CD4 and the CD4 binding site antibody 654-30D. It showed low binding to the monoclonal antibody 654- 30D, moderate binding to 2G12, Chessie 13-39.1 and 9301, and high binding to ID6, but did not show binding to any of the other antibodies used in the recognition profile. Gp120 from the CCR5-using isolate, FV5, showed low binding to the monoclonal antibodies F425 B4a1 and Chessie 13-39.1, moderate binding to 2G12, and showed good binding to 9301and ID6. FV5 gp120 could not, however, bind to CD4. This is likely to be related to a D368G substitution, a mutation affecting a critical structural determinant of CD4 binding. The lack of CD4-binding activity of this gp120 highlights the importance of Asp368 for CD4 binding and hints at a region vulnerable for therapeutic targeting. Our results also highlight the challenges of developing broadly therapeutic drugs for HIV-1, as well as the importance of investigating the specific biochemical and pathogenic properties associated with subtype C HIV-1. en
dc.language.iso en en
dc.subject HIV-1 subtype C gp120 en
dc.title The expression, purification and characterisation of recombinant HIV-1 subtype C gp120 en
dc.type Thesis en


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