Cloning, DNA sequencing and expression of (3-17)beta hydroxysteroid dehydrogenase from Pseudomonas testosteroni

We describe the cloning, sequencing and overexpression of the (3-17)beta hydroxysteroid dehydrogenase gene of Pseudomonas testosteroni. A genomic library of Ps. testosteroni total DNA constructed from SauIIIA digests ligated to a lambda gt11 vector was probed with polyclonal antibody raised against purified enzyme. Subclones derived from a recombinant phage containing a 2661-base-pair insert were sequenced and found to contain an open reading frame of 765 base pairs that corresponds to a protein of 254 amino acid residues. A 1492-base-pair fragment was inserted into pBR322 plasmid vector and used to construct a strain of E. coli HB101 that overexpressed the steroid dehydrogenase gene.