Previous experiments have shown that substitutions of some amino acids in a conserved region of the vaccinia virus DNA polymerase are allowed. Vaccinia virus containing the altered polymerase formed plaques similar to those of wild-type virus. To ultimately identify what aspect of DNA polymerase function was associated with the structure of this particular conserved region, an attempt was made to express the enzyme in bacteria. If modest amounts of functional enzyme can be expressed from a cloned gene, then the in vivo substitutions can be tested in vitro. Initially, about one-half of the polymerase gene, from the carboxy end, was cloned into the pET16b expression vector. This construct produced moderate amounts of a 56 kD protein after induction of bacterial cultures. The protein was insoluble, but this result was satisfactory, since it protected the protein from degradation. Vigorous isolation procedures showed that the target protein was in large inclusion bodies. To solubilize the protein, these bodies were blended at high speed in 6 M guanidine chloride and 100 mM dithiothreitol and then heated at 60 degrees C for 60 minutes. The dissolved protein was fractionated on a sizing column in 6 M guanidine, and the 56 kD protein was collected and precipitated with ethanol. This protein was used as an antigen which will, hopefully, produce an antibody that will allow purification of polymerase from crude extracts. Also, to produce alterations of the entire DNA polymerase gene, I cloned the entire gene in M13mp19. This system produces a phage that contains a recombinant DNA with one strand of the gene. This construct allows the application of a modified form of the Eckstein mutagenesis system, in which the in vitro mutagenic manipulations can be completed in one day. This application depends upon the action of T5 D15 exonuclease. This enzyme has been tested and shown to degrade circular single-stranded DNA and linear and nicked circle duplex DNA. Since tests also show that closed circular DNA is unaffected, homoduplexes of altered DNA produced by the Eckstein system can be prepared from microgram amounts of the original template.
