Purification of crystallizable recombinant SIVmac251-32H proteinase.

Important note: Information in this article was accurate in 1994. The state of the art may have changed since the publication date.

Purification of crystallizable recombinant SIVmac251-32H proteinase.

Protein Expr Purif. 1994 Feb;5(1):76-83. Unique Identifier : AIDSLINE MED/94220860
Sugrue RJ; Almond N; Kitchin P; Richardson SM; Wilderspin AF; Department of Crystallography, Birkbeck College, London, United; Kingdom.

Abstract: We have cloned a simian immunodeficiency virus (SIV) proteinase gene directly from proviral DNA of the infectious viral stock SIVmac251-32H (11/88 pool). The deduced amino acid sequence from this proteinase gene is similar to that for the published SIVmac239 molecular clone. SIVmac251-32H proteinase (SIV PR) and its flanking pol sequences were expressed in Escherichia coli as a fusion protein with most of the T7 bacteriophage gene 10 protein. The expressed protein formed cytoplasmic inclusion bodies which were solubilized in 8 M urea, and the recombinant SIV PR was refolded, yielding active, self-processed enzyme. The SIV PR was purified to homogeneity using a single pepstatin A affinity chromatography step, and had a specific peptidolytic activity of 20 mumol/min/mg. Enzymatic characteristics similar to those previously documented for other immunodeficiency virus proteinases (EC 3.4.23) were observed. These include an acidic pH optimum (pH 5.3), sensitivity to sodium chloride concentration, and complete inhibition by pepstatin A. In addition to these properties we have observed quantitative crystallization from low protein concentrations. We describe the first crystal habit for the proteinase from the HIV-2/SIV class of immunodeficiency virus, which is distinctly different from that for HIV-1 proteinase crystals.

Keywords: Amino Acid Sequence Aspartic Proteinases/BIOSYNTHESIS/*ISOLATION & PURIF Base Sequence Chromatography, Affinity Crystallization Escherichia coli Molecular Sequence Data Pepstatins Protein Folding Recombinant Fusion Proteins/BIOSYNTHESIS/*ISOLATION & PURIF Support, Non-U.S. Gov't SIV/*ENZYMOLOGY/GENETICS Urea JOURNAL ARTICLE
940830
M9480753

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