Cloning, sequencing and expression of Pseudomonas testosteroni gene encoding 3α-hydroxysteroid dehydrogenase

• Published in: The Journal of steroid biochemistry and molecular biology 1995-11, Vol.55 (2), p.233-238
• Main Authors: Abalain, J.H., Di Stefano, S., Abalain-Colloc, M.L., Floch, H.H.
• Format: Article
• Language: English
• Subjects: 3-Hydroxysteroid Dehydrogenases - biosynthesis; 3-Hydroxysteroid Dehydrogenases - genetics; 3-Hydroxysteroid Dehydrogenases - isolation & purification; Amino Acid Sequence; Base Sequence; Biological and medical sciences; Cloning, Molecular; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; Escherichia coli; Fundamental and applied biological sciences. Psychology; Gene Expression; Genes, Bacterial; Genes. Genome; Genomic Library; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Plasmids; Pseudomonas - enzymology; Pseudomonas - genetics; Pseudomonas testosteroni; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Restriction Mapping
• ISSN: 0960-0760; 1879-1220
• DOI: 10.1016/0960-0760(95)00170-5

We describe the cloning, sequencing and expression of the 3α-hydroxysteroid dehydrogenase (3α-HSD) gene of Pseudomonas testosteroni. A genomic library of P. testosteroni total DNA constructed from SauIIIA digests ligated to an λgt11 vector was probed with a polyclonal antibody raised against purified enzyme. Subclones derived from a recombinant phage containing a 1746 bp insert were sequenced and found to contain an open reading frame of 696 bp that corresponds to a protein of 231 amino acid residues. A search for homologous proteins was performed. No similarity was observed when comparing 3α-HSD with known members of the short-chain dehydrogenase family. However a small proteic fragment (80 amino acids) shows homology with the N-terminal sequence of bacterial L7 L12 ribosomal proteins.