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Overexpression and purification of enzymatically active recombinant integrase protein of Rous sarcoma virus.

Authors
  • Marczinovits, I
  • Molnár, J
  • Sóki, J
  • Fodor, I
Type
Published Article
Journal
Virus genes
Publication Date
Aug 01, 1992
Volume
6
Issue
3
Pages
301–306
Identifiers
PMID: 1329343
Source
Medline
License
Unknown

Abstract

The carboxy-terminal domain of polymerase gene of Rous sarcoma virus was cloned into an expression vector under the control of lac regulatory elements, resulting in the plasmid pMF1413. Upon isopropyl-beta-D-thiogalactopyranoside induction, viral integration (IN) protein was expressed in large quantity in Escherichia coli. The expressed recombinant protein was prepurified by successive washing of the bacterial pellet with 0.1 M NaCl and detergents. Further purification was performed in high yield by standard chromatography methods. The purified enzyme revealed selective DNA cleaving activity on supercoiled plasmid with the LTR-LTR junction fragment. The reaction was metal ion dependent, with a preference for Mn2+ over Mg2+, and showed substrate specificity at 1 mM MnCl2.

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