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The reverse transcriptase (RT) of HIV is an important target for chemotherapy as demonstrated by the effective treatment of AIDS patients with zidovudine, a potent inhibitor of RT. Structural studies of HIV RT were therefore undertaken with a view to designing more effective inhibitors. To obtain sufficient quantities of enzyme for these studies the reverse transcriptase gene of HIV was cloned into a high level expression plasmid yielding reverse transcriptase at a level of 10% of the total Escherichia coli proteins. Monoclonal antibodies to RT were raised in mice and have been used to purify the enzyme by immunoaffinity chromatography. Crystallization of the enzyme has been achieved and studies are underway to determine its three-dimensional structure. In addition, carboxy-terminal truncated mutants were prepared by inserting stop codons into the gene at appropriate sites. The proteins expressed were analysed for RT and RNase H activity and used for mapping RT epitopes. This, together with previous data on site-directed mutagenesis of conserved regions of HIV RT has helped to map some of the structural and functional regions of the enzyme.

Original publication

DOI

10.1093/jac/23.suppl_a.47

Type

Journal article

Journal

J Antimicrob Chemother

Publication Date

01/1989

Volume

23 Suppl A

Pages

47 - 54

Keywords

Animals, Antibodies, Monoclonal, Antiviral Agents, Cloning, Molecular, Drug Design, Epitopes, HIV, Humans, Mice, Peptide Mapping, RNA-Directed DNA Polymerase, Recombinant Proteins, Reverse Transcriptase Inhibitors