Cloning, Expression, and Characterization of Family A DNA Polymerase from Massilia aurea

Mau DNA polymerase is a family A DNA polymerase isolated from Massilia aurea. In this study, a recombinant plasmid, His(6)-tagged Mau-pET28c, was constructed. His-tagged Mau was expressed in Escherichia coli Rosseta 2 (DE3) competent cells and, after optimization of purification conditions, was successfully isolated via a two-step purification system by Ni2+-chelating affinity chromatography followed by heparin affinity chromatography. The biochemical properties of Mau DNA polymerase were investigated next. This polymerase showed maximal polymerase activity at 30 & DEG;C, pH 8.4-8.8, 2-10 mM MgCl2, and 10-40 mM KCl. Kinetic parameters of correct and incorrect dNTP incorporation as well as DNA-binding affinity were determined too. K-d,app(dNTP) values were found to be 16 & mu;M for correct dNTP and 200-500 & mu;M for incorrect dNTP. The kinetic parameter k(cat) turned out to be 0.2 s(-1) for correct dNTP incorporation and an order of magnitude less for incorrect dNTP incorporation. It was demonstrated that Mau DNA polymerase has 5 & PRIME;& RARR;3 & PRIME; and 3 & PRIME;& RARR;5 & PRIME; exonuclease activities associated with the main activity.