The goal was to insert the vaccinia virus DNA polymerase gene into vaccinia virus vT7lacOI that can overexpress a cloned gene in infected cells without any additional virus or plasmid. First, the entire gene was inserted into the DNA of the replicative form of M13mp19 bacteriophage. This construct facilitates gene modification by the Eckstein mutagenesis procedure which was used to install a unique NdeI site at the locus of the first codon for the polymerase. The modified gene was inserted into the new vaccinia virus transfer vector pVote2 which targets the polymerase gene into the hemagglutinin region of the vT7lacOI virus that encodes the T7 RNA polymerase. Since the DNA polymerase gene is linked to a T7 promotor after insertion into the transfer vector, it is actively transcribed by the T7 polymerase after induction of infected cells. Recombinant viruses, which were selected by resistance to mycophenolic acid, produced very small plaques, which frequently did not survive successive passage. Of the 320 plaques tested only two were able to express a recombinant protein (RP) of the size of the DNA polymerase after induction of infected cells. For one recombinant the relative yield of the induced protein produced 15 to 19 hours post-infection was estimated as twenty per cent of the soluble protein. Small amounts of radiolabeled RP were purified by a modification of a standard procedure. Chromatographic patterns of RP on a sizing column and DEAE were similar to that for the normal enzyme but the affinity of RP for heparin was less than expected. One disadvantage of this expression system is that the low yield of recombinant virus precludes the rapid accumulation of virus stock. Initially it was difficult to insert DNA segments with an NdeI cohesive end and a blunt end into pVote2. This vector was modified to facilitate insertion of such segments by introducing a second blunt end site in the cloning region at a position removed from the Nde I site.
