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Title
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Purification of crystallizable recombinant SIVmac251-32H proteinase.
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Authors
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R.J.Sugrue,
N.Almond,
P.Kitchin,
S.M.Richardson,
A.F.Wilderspin.
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Ref.
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Protein Expr Purif, 1994,
5,
76-83.
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PubMed id
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Abstract
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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.
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