Molecular cloning and expression of the beta-hydroxysteroid dehydrogenase gene from Pseudomonas testosteroni.
The structural gene (hsd) of the Pseudomonas testosteroni encoding the 17 beta-hydroxysteroid dehydrogenase has been cloned using the cosmid vector pVK102. 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]methionine-labeled polypeptides.
Genti-Raimondi
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Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Spain.
Tolmasky
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ME
Patrito
L C
LC
Flury
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A
Actis
L A
LA
D015067:17-Hydroxysteroid Dehydrogenases / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D003360:Cosmids / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D015698:Genomic Library; D015151:Immunoblotting; D016297:Mutagenesis, Site-Directed; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D013739:Testosterone / Q000378:metabolism
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013739:Testosterone; D015067:17-Hydroxysteroid Dehydrogenases; C032690:3-alpha-(17-beta)-hydroxysteroid dehydrogenase (NAD(+))