Document Detail


Heterologous expression and purification of Vibrio proteolyticus (Aeromonas proteolytica) aminopeptidase: a rapid protocol.
MedLine Citation:
PMID:  19233285     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Mariam Hartley; Wei Yong; Brian Bennett
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2009-02-20
Journal Detail:
Title:  Protein expression and purification     Volume:  66     ISSN:  1096-0279     ISO Abbreviation:  Protein Expr. Purif.     Publication Date:  2009 Jul 
Date Detail:
Created Date:  2009-04-13     Completed Date:  2009-06-02     Revised Date:  2014-03-19    
Medline Journal Info:
Nlm Unique ID:  9101496     Medline TA:  Protein Expr Purif     Country:  United States    
Other Details:
Languages:  eng     Pagination:  91-101     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Aeromonas / enzymology*
Aminopeptidases / genetics,  isolation & purification*,  metabolism*
Bacterial Proteins / genetics,  isolation & purification*,  metabolism*
Crystallography
Isoenzymes / genetics,  isolation & purification*,  metabolism*
Molecular Sequence Data
X-Ray Diffraction
Grant Support
ID/Acronym/Agency:
R01 AI 056231/AI/NIAID NIH HHS; R01 AI056231/AI/NIAID NIH HHS; R01 AI056231-01/AI/NIAID NIH HHS; R01 AI056231-02/AI/NIAID NIH HHS; R01 AI056231-03/AI/NIAID NIH HHS; R01 AI056231-04/AI/NIAID NIH HHS; R01 AI056231-05/AI/NIAID NIH HHS
Chemical
Reg. No./Substance:
0/Bacterial Proteins; 0/Isoenzymes; EC 3.4.11.-/Aminopeptidases; EC 3.4.11.10/bacterial leucyl aminopeptidase
Comments/Corrections
Erratum In:
Protein Expr Purif. 2010 Apr;70(2):299
Note: Yong, Wei [added]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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