Purification and properties of Aquifex aeolicus DNA polymerase expressed in Escherichia coli

Abstract

The gene encoding Aquifex aeolicus ( Aae) DNA polymerase was expressed under the control of the trp promoter on a high-copy plasmid, pTRPNS, in Escherichia coli. The expressed enzyme was purified 11-fold with a 13.8% yield and a specific activity of 2268.3 U mg −1. The optimum pH of the enzyme was 6.8–7.2. The optimal concentrations of KCl and Mg 2+ were 20–30 mM and 4–5 mM, respectively. Aae DNA polymerase contained a double-strand-dependent 3′→5′ proofreading exonuclease activity but lacked any detectable 5′→3′ exonuclease activity.