Cloning of the gene for delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni

We have cloned an approx. 5-kb fragment of Pseudomonas testosteroni DNA containing the structural gene of delta 5-3-ketosteroid isomerase into the EcoRI site of the lambda gt11 genome. Escherichia coli infected with these recombinant phages produce a polypeptide which is recognized by antiserum raised against the purified isomerase. Four of the recombinant lambda gt11 clones contain significant levels of isomerase activity and produce an immunopositive polypeptide of the same apparent Mr as the native isomerase obtained from P. testosteroni. The approx. 5-kb fragment hybridizes to synthetic 21-mer and 17-mer oligodeoxynucleotide mixtures corresponding to the 5' and 3' regions, respectively, of the expected nucleotide sequence of the gene.