CONSTRUCTION AND EXPRESSION OF A MODULAR GENE ENCODING BACTERIOPHAGE-T7 RNA-POLYMERASE

A modular gene that encodes T7 RNA polymerase (T7 RNAP) and consists o f cassettes delimited by unique restriction sites was constructed. The modular and wild-type genes of T7 RNAP were cloned into a vector desi gned to express His-tagged proteins. The modular and wild-type genes p rovided the same level of protein expression (i.e., T7 RNAP represente d up to 30% of the total protein in Escherichia coli strain BL21). Pur ification of both proteins by immobilized metal ion affinity chromatog raphy (IMAC) resulted in similar yields (700-800 mu g of enzyme per 20 ml of culture) and purity (>95%) as indicated by Coomassie blue stain ing, Western blotting and the absence of detectable contaminating nucl ease activities. Both proteins exhibited identical efficiency in trans cription assays, and their specific activities (about 200 U/mu g) were close to that of a commercial T7 RNAP preparation. The modular gene p rovides a useful tool for cassette directed mutagenesis of T7 RNAP. (C ) 1997 Elsevier Science B.V.