       Document 0145
 DOCN  M9460145
 TI    Purification of crystallizable recombinant SIVmac251-32H proteinase.
 DT    9408
 AU    Sugrue RJ; Almond N; Kitchin P; Richardson SM; Wilderspin AF; Department
       of Crystallography, Birkbeck College, London, United; Kingdom.
 SO    Protein Expr Purif. 1994 Feb;5(1):76-83. Unique Identifier : AIDSLINE
       MED/94220860
 AB    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.
 DE    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

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

