OVEREXPRESSION AND PURIFICATION OF THE VACCINIA VIRUS-DNA POLYMERASE

We have overexpressed the vaccinia virus DNA polymerase using the hybr id vaccinia virus/T7 expression system. Accumulation of the DNA polyme rase to levels as high as 10% of the total protein was observed follow ing coinfection of BSC40 cells with the appropriate vaccinia recombina nts. Although the DNA polymerase produced at 37-degrees-C was largely insoluble, 25% of the recombinant protein could be recovered as solubl e protein when infected cultures were maintained at 32-degrees-C. Star ting with cytoplasmic lysates of coinfected cells, a rapid and reprodu cible purification protocol that yielded apparently homogeneous prepar ations of the DNA polymerase after four chromatographic steps was esta blished. Typically, 0.3 mg of purified DNA polymerase was obtained fro m 27 mg of total protein within 10 h after harvesting infected cells. As was previously described for the DNA polymerase purified from vacci nia-infected cells (Challberg and Englund, J. Biol. Chem., 254, 7812-7 819,1979), the purified recombinant enzyme displayed both polymerase a nd 3'-5' exonuclease activities but lacked detectable 5'-3' exonucleas e activity. Kinetic analysis of nucleotide incorporation catalyzed by the vaccinia enzyme revealed apparent K(m) values of 0.9, 2.9, 4.0, an d 2.7 muM for dGTP, dATP, TTP, and dCTP, respectively. (C) 1994 Academ ic Press, Inc.