Abstract
The structural gene ( hsd) of the Pseudomonas testosteroni encoding the 17β-hydroxy steroid dehydrogenase has been cloned using the cosmid vector pVK.102. Escherichia coli carrying recombinant clones of hsd, isolated by immunological screening, were able to express the biologically active enzyme, as measured by the conversion of testosterone into androstenedione. Subcloning experiments, restriction and deletion analysis, and site-directed insertion mutagenesis showed that the hsd gene is located within a 1.3-kb HindIII- PstI restriction fragment. A 26.5-kDa protein encoded by a recombinant plasmid containing this Ps. testosteroni DNA restriction fragment was detected by SDS-PAGE analysis of in vitro [ 35S]methioninelabeled polypeptides.
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