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PDBsum entry 3fh4
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References listed in PDB file
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Key reference
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Title
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Heterologous expression and purification of vibrio proteolyticus (aeromonas proteolytica) aminopeptidase: a rapid protocol.
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Authors
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M.Hartley,
W.Yong,
B.Bennett.
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Ref.
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Protein Expr Purif, 2009,
66,
91.
[DOI no: ]
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PubMed id
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Abstract
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Metalloaminopeptidases (mAPs) are enzymes that are involved in HIV infectivity,
tumor growth and metastasis, angiogenesis, and bacterial infection.
Investigation of structure-function relationships in mAPs is a prerequisite to
rational design of anti-mAP chemotherapeutics. The most intensively studied
member of the biomedically important dinuclear mAPs is the prototypical secreted
Vibrio proteolyticus di-zinc aminopeptidase (VpAP). The wild-type enzyme is
readily purified from the supernatant of cultures of V. proteolyticus, but
recombinant variants require expression in Escherichia coli. A greatly improved
system for the purification of recombinant VpAP is described. A VpAP-(His)(6)
polypeptide, containing an N-terminal propeptide, and a C-terminal (His)(6)
adduct, was purified by metal ion affinity chromatography from the supernatant
of cultures of E. coli. This single step replaced the sequence of
(NH(4))(2)SO(4) fractionation, and anion-exchange and hydrophobic interaction
chromatographic separations of earlier methods. Traditionally, recombinant VpAP
proenzyme has been treated with proteinase K and with heat (70 degrees C), to
remove the N- and C-terminal regions, and yield the mature active enzyme. This
method is unsuitable for VpAP variants that are unstable towards these
treatments. In the new method, the hitherto noted, but not fully appreciated,
ability of VpAP to autocatalyze the hydrolysis of the N-terminal propeptide and
C-terminal regions was exploited; extensive dialysis of the highly purified
VpAP-(His)(6) full-length polypeptide yielded the mature active protein without
recourse to proteinase K or heat treatment. Purification of variants that have
previously defied isolation as mature forms of the protein was thus carried out.
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