Abstract
A new method for the complete purification of bacteriophage Qβ RNA-dependent RNA polymerase is described. A Qβ strain containing an amber mutation in the coat cistron is used to achieve enzyme overproduction and non-lytic cell growth. Enzyme activity is followed by a host factor-independent specific assay for replicase—the rifampicin-resistant synthesis of poly(G) on poly(C) templates. By step-wise liquid polymer phase partitioning, DEAE-cellulose and phosphocellulose column chromatography, followed by high salt glycerol gradient centrifugation, approx. 10 mg of enzyme more than 95 % pure is obtained from 250 g of mutant infected Escherichia coli.
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