CONSTRUCTION OF AN EXPRESSION AND SITE-DIRECTED MUTAGENESIS SYSTEM OF HALOALKANE DEHALOGENASE IN ESCHERICHIA-COLI

Haloalkane dehalogenase from Xanthobacter autotrophicus was efficiently expressed in Escherichia coli BL21(DE3) and E. coli JM101. After introduction of restriction sites by PCR the haloalkane dehalogenase gene (dhLA) was translationally fused behind the T7 (φ10), trc, and tac promoters. This resulted in expression at 30°C up to 38 and 18% of the total soluble cellular protein with the T7 and trc promoters, respectively. Dehalogenase expression under control of the tac promoter was below 1% of the soluble cell protein, however. Aggregation of haloalkane dehalogenase into inclusion bodies was found during growth at 37°C but not at 30°C. Aggregates were also formed from intact enzyme upon incubation at 37°C of cells or crude extracts containing active mature dehalogenase. The high level of expression resulted in a short purification procedure in which 30-35 mg highly enriched haloalkane dehalogenase was obtained from an 0.5 l culture. For the production of single-stranded DNA an f1(+) origin was introduced in the T7 expression system. © 1993 Academic Press. All rights reserved.