RAPID MUTAGENESIS AND PURIFICATION OF PHAGE RNA-POLYMERASES

We have developed plasmid-based expression systems that encode modifie d forms of T7 RNA polymerase (RNAP) having 6-12 histidine residues fus ed to the amino terminus. The histidine-tagged RNAPs (His-T7 RNAPS) ar e indistinguishable from the wild-type (WT) enzyme in nearly all bioch emical assays. Similar plasmids that encode His-tagged T3 and SP6 RNAP s have also been constructed. To facilitate site-directed mutagenesis of the RNAP gene, the size of the target plasmid was minimized by usin g T7 RNAP itself as a selectable marker. BL21 (DCAT4) cells (which car ry a chromosomal copy of the chloramphenicol acetyl-transferase cat ge ne under control of a T7 promoter) are resistant to chloramphenicol wh en functional T7 RNAP is expressed, thus allowing the selection and ma intenance of the target plasmid in these cells. Mutagenesis is accompl ished by denaturing the plasmid, annealing mutagenic DNA primers, and repairing the plasmid with T4 DNA polymerase. Two DNA primers are used : one corrects a defect in the bla gene, the other introduces the desi red mutation into the RNAP gene; 30-85% of the ampicillin-resistant tr ansformants carry the desired mutation in the RNAP gene. By using BL21 (DCAT4) cells as a recipient for transformation the functional integr ity of the RNAP gene may conveniently be monitored by assessing the le vel of chloramphenicol resistance in vivo. Methods for rapid, simultan eous purification of multiple samples of modified (His-tagged) and con ventional RNAPs are described. Together, these developments greatly en hance our ability to characterize this important class of enzymes. (C) 1997 Academic Press.