Pharmacia Corporation

Mark C Walker

John S Ng

Alan Michael Easton

Ping T Wang

Robert Clayton

Dean M Messing

Suzanne L Bolten

Beverly Reitz

Leslie C Engel

The present invention relates to a novel cytochrome P450-like enzyme (11 alpha hydroxylase) and an oxidoreductase (oxidoreductase) isolated from cDNA library generated from the mRNA ofspores. When the cDNA encoding the 11 alpha hydroxylase was co-expressed in(Sf-9) insect cells with the cDNA encoding human oxidoreductase as an electron donor, it successfully catalyzed the conversion of the steroid substrate 4-androstene-3,17-dione (AD) to 11 alpha-hydroxy-AD as determined by HPLC analysis. The invention also relates to nucleic acid molecules associated with or derived from these cDNAs including complements, homologues and fragments thereof, and methods of using these nucleic acid molecules, to generate, for example, polypeptides and fragments thereof. The invention also relates to the generation of antibodies that recognizes the11 alpha hydroxylase and oxidoreductase and methods of using these antibodies to detect the presence of these native and recombinant polypeptides within unmodified and transformed host cells, respectively. The invention also provides methods of expressing the11 alpha hydroxylase gene separately, or in combination with human oroxidoreductase, in heterologous host cells, to facilitate the bioconversion of steroid substrates to their 11 alpha hydroxy-counterparts.

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Aspergillus ochraceus 11 alpha hydroxylase and oxidoreductase

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Chesterfield, MO, United States of America

Mark C Walker