6th International AIDS Conference


San Francisco, California, USA — June 20-23, 1990

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Biochemical characterization of purified recombinant HIV-1 REV protein.

Int Conf AIDS 1990 Jun 20-23; 6:321 (abstract no. 1031)
Herlihy WC, Daly T, Donner A, Maione T, Frankel A, Cullen B, Auer M, Rusche J, Doten R; Repligen Corporation, Cambridge, MA, USA

OBJECTIVE: Develop methods for the production of large amounts of highly purified HIV-1 REV protein. Characterize the physical properties of wild type and mutant proteins to aid in understanding the mechanism by which REV alters gene expression and aid in designing antiviral compounds.

METHODS: The REV gene was cloned as an operon fusion with the B-glucuronidase gene of E. coli and purified to homogeneity. The wild type and mutant proteins were characterized regarding solubility, melting temperature, RNA binding kinetics and proteolytic degradation patterns using a variety of techniques.

RESULTS: REV protein and two mutant proteins were purified and characterized. The wild type protein is maximally soluble below pH 5 and above pH 9. Solubility is enhanced at pH 7.0 by increasing ionic strength or the addition of RNA. Circular dichroism measurements were used to estimate the alpha helix content and the TM of this ordered structure at various pH. The REV protein binds RRE containing RNA with a T 1/2 of 20 minutes. The affinity and stoichiometry of wild type and mutant protein will be presented. Comparative proteolytic profiles of the REV proteins will also be presented.

CONCLUSIONS: Biologically active REV protein can be purified to homogeneity in gram amounts from E. coli. Physical analysis of REV and comparison with mutant REV proteins will illuminate the location and shape of REV active sites. This should allow for the rapid design and analysis of antiviral compounds.

Keywords: AEGIS, Gene Products, rev, HIV-1, Genes, rev, Recombinant Proteins, RNA, Binding Sites, Circular Dichroism, RNA, Antisense, Escherichia coli, Gene Expression Regulation, Viral, Mutation, Kinetics, RNA Probes, Gene Expression, genetics, ICA6KWDaegis,geneproducts,rev,hiv-1,genes,rev,recombinantproteins,rna,bindingsites,circulardichroism,rna,antisense,escherichiacoli,geneexpressionregulation,viral,mutation,kinetics,rnaprobes,geneexpression,genetics,ica6

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