Human immunodeficiency virus (HIV) and its gradual weakening of the immune system is an ever growing threat. Acquired immune deficiency syndrome (AIDS), the final stage of HIV, renders a person vulnerable to various opportunistic infections, which in the end lead to death. Apart from intensive vaccine studies, treatment research mainly focuses on preventing the individual HIV enzymes (reverse transcriptase, integrase and protease) from performing their functions. Entry inhibitors, however, block viral entry into the cell, while antisense drugs lock onto the viral genome to keep it from functioning. In this study production of active recombinant HIV-1 protease and integrase was attempted for future drug screening programs. HIV-1 protease was cloned into a pET28b(+) vector and expressed in ROSETTA(DE3)pLysS cells. The protein was purified using a nickel-affinity column utilizing the hexa-histidine tag encoded by the vector. Gel filtration chromatography was attempted after refolding of the protease, but protease yield seemed to decrease with the additional purification step. Partially purified protease was characterized with kinetic studies. Kinetic parameters of HIV-1 protease were determined to be Km = 592 μM, Vmax = 0.59 μM/min and kcat = 31 s-1. HIV-1 integrase, which was cloned into a pET15b vector, was expressed in E. coli BL21(DE3) cells. The coding sequence had been mutated to introduce the amino acid substitutions F185K and C280S, increasing solubility of the protein. The first step in purification of this protein was nickel-affinity chromatography, after which cation exchange chromatography was attempted. HIV-1 integrase concentration was low throughout experiments and no clear elution from the cation exchange column could be observed. A non-radioactive enzyme linked HIV-1 integrase assay failed to detect integrase activity. Modifications to future studies of the integrase are suggested in the chapter involved.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:nmmu/vital:10325 |
| Date | January 2009 |
| Creators | Bosch, Janine |
| Publisher | Nelson Mandela Metropolitan University, Faculty of Science |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis, Masters, MSc |
| Format | x, 133 leaves, pdf |
| Rights | Nelson Mandela Metropolitan University |
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