Purification of crystallizable recombinant SIVmac251-32H proteinase.
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.
Sugrue
R J
RJ
Department of Crystallography, Birkbeck College, London, United Kingdom.
Almond
N
N
Kitchin
P
P
Richardson
S M
SM
Wilderspin
A F
AF
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000096:biosynthesis / Q000302:isolation & purification*; D001483:Base Sequence; D002846:Chromatography, Affinity; D003460:Crystallization; D004926:Escherichia coli; D008969:Molecular Sequence Data; D010436:Pepstatins; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D015302:Simian Immunodeficiency Virus / Q000201:enzymology* / Q000235:genetics; D014508:Urea
D010436:Pepstatins; D011993:Recombinant Fusion Proteins; C014195:Streptomyces pepsin inhibitor; D014508:Urea; D016282:Aspartic Acid Endopeptidases; C077164:SIV(mac) proteinase; C031375:pepstatin