6th International AIDS Conference


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

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Hydrolysis of insoluble polyprotein substrate with two identical cleavage sites by recombinant HIV-1 protease.

Int Conf AIDS 1990 Jun 20-23; 6:324 (abstract no. 1041)
Tarasova NI, Gulnik SV, Shulenin SV, Bobkov AF, Garaev MM; Moscow State University, Department of Chemistry, USSR

OBJECTIVE: The main goal of the present work was the investigation of the expression of HIV-encoded protease in E.coli and of enzymatic properties of the recombinant enzyme.

METHODS: A hybrid plasmid pPR6 coding the HIV sequence from Asp-5 to Ile-868 of the pol open reading frame has been expressed in E.coli. As a substrate a fusion-polypeptide of beta-galactosidase with a doubled sequence of a part of p55 gag protein, containing a site of HIV-protease action situated on a border of p17 and p24 proteins has been used.

RESULTS: During the expression of pol gene product an autoprocessing was observed leading to formation of active protease and reverse transcriptase. The protease was purified to homogeneity. pH 6-7 was determined to be optimal for the enzyme action. Maximal stability was observed between pH 4 and 5, Substrate hydrolysis was completely inhibited by Cu++ and Zn++ at concentrations of 10mM and 2mM respectively.

CONCLUSIONS: The E.coli-produced HIV protease will be used in search for inhibitors and to facilitate structural and biochemical studies. The model suggested can be used as a convenient test system for detection of HIV-protease activity.

Keywords: AEGIS, HIV Protease, Gene Products, gag, Gene Products, pol, Hydrolysis, RNA-Directed DNA Polymerase, Plasmids, Endopeptidases, Escherichia coli, HIV, Hydrogen-Ion Concentration, ICA6KWDaegis,hivprotease,geneproducts,gag,geneproducts,pol,hydrolysis,rna-directeddnapolymerase,plasmids,endopeptidases,escherichiacoli,hiv,hydrogen-ionconcentration,ica6

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