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

摘要

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.