Overexpression and purification of the vaccinia virus DNA polymerase. Protein Expr Purif 1994 Aug;5(4):409-21
Date
08/01/1994Pubmed ID
7950389DOI
10.1006/prep.1994.1059Scopus ID
2-s2.0-0028484045 (requires institutional sign-in at Scopus site) 34 CitationsAbstract
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.
Author List
McDonald WF, Traktman PMESH terms used to index this publication - Major topics in bold
AnimalsBacteriophage T7
Base Sequence
Blotting, Southern
Cells, Cultured
DNA-Directed DNA Polymerase
Exodeoxyribonuclease V
Exodeoxyribonucleases
Genetic Vectors
Mice
Molecular Sequence Data
Nucleotides
Recombinant Proteins
Substrate Specificity
Vaccinia virus
