Overexpression and purification of the vaccinia virus DNA polymerase.
We have overexpressed the vaccinia virus DNA polymerase using the hybrid vaccinia virus/T7 expression system. Accumulation of the DNA polymerase to levels as high as 10% of the total protein was observed following coinfection of BSC40 cells with the appropriate vaccinia recombinants. Although the DNA polymerase produced at 37 degrees C was largely insoluble, 25% of the recombinant protein could be recovered as soluble protein when infected cultures were maintained at 32 degrees C. Starting with cytoplasmic lysates of coinfected cells, a rapid and reproducible purification protocol that yielded apparently homogeneous preparations of the DNA polymerase after four chromatographic steps was established. Typically, 0.3 mg of purified DNA polymerase was obtained from 27 mg of total protein within 10 h after harvesting infected cells. As was previously described for the DNA polymerase purified from vaccinia-infected cells (Challberg and Englund, J. Biol. Chem., 254, 7812-7819, 1979), the purified recombinant enzyme displayed both polymerase and 3'-5' exonuclease activities but lacked detectable 5'-3' exonuclease activity. Kinetic analysis of nucleotide incorporation catalyzed by the vaccinia enzyme revealed apparent Km values of 0.9, 2.9, 4.0, and 2.7 microM for dGTP, dATP, TTP, and dCTP, respectively.
McDonald
W F
WF
Department of Microbiology, Cornell University Medical College, New York, New York 10021.
Traktman
P
P
D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D002478:Cells, Cultured; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000032:analysis; D005822:Genetic Vectors; D051379:Mice; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013379:Substrate Specificity; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics
D009711:Nucleotides; D011994:Recombinant Proteins; C090592:DNA polymerase, vaccinia virus; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V