Expression in Escherichia coli of a rat cDNA encoding an apurinic/apyrimidinic endonuclease

Ikramul Huq, Teresa M. Wilson, Mark R. Kelley, Walter A. Deutsch

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

A rat cDNA (rAPEN) with 85% DNA identity to the major human apurinic/apyrimidinic (AP) endonuclease gene was used to construct a fusion between it and glutathione-S-transferase (GST). The GST-rAPEN fusion was subsequently overexpressed in Escherichia coli, purified on glutathione-agarose affinity columns, and the purified protein tested for AP endonuclease activity. DNA nicks were found to be specifically introduced into AP DNA in a reaction that was dependent upon the time of incubation and the amount of GST-rAPEN added. The DNA scissions produced by GST-rAPEN were determined to be adjacent and 5′ to an AP site. The purified fusion protein was also able to efficiently remove 3′-(4 hydroxy-5-phospho-2-pentenal) residues, and to a lesser extent 3′-phosphoglycolate residues. The GST-rAPEN activity failed to exhibit any 3′-5′ exonuclease activity, a characteristic shared by the major AP endonuclease in bovine and human.

Original languageEnglish (US)
Pages (from-to)191-199
Number of pages9
JournalMutation Research-DNA Repair
Volume337
Issue number3
DOIs
StatePublished - Nov 1995

Keywords

  • cDNA expression

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology
  • Toxicology

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