Document Type

Article

Language

eng

Publication Date

7-2009

Publisher

Elsevier

Source Publication

Protein Expression and Purification

Source ISSN

1046-5928

Original Item ID

DOI: 10.1016/j.pep.2009.02.011

Abstract

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 (NH4)2SO4 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 °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.

Comments

Accepted version. Protein Expression and Purification, Vol. 66, No. 1 (July 2009): 91-101. DOI. © 2009 Elsevier. Used with permission.

NOTICE: this is the author’s version of a work that was accepted for publication in Protein Expression and Purification. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Protein Expression and Purification, VOL 66, ISSUE 1, July 2009, DOI.

Brian Bennett was affiliated with the Medical College of Wisconsin at the time of publication.

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