Cloning and expression of cDNA encoding a bovine adrenal cytochrome P-450 specific for steroid 21-hydroxylation

We isolated a cDNA clone encoding a bovine adrenal cytochrome P-450 specific for steroid 21-hydroxylation (P-450(C21)). Serum from rabbits immunized with purified P-450(C21) precipitated a single protein from the products of an in vitro translation reaction using bovine adrenal mRNA. This protein migrated with P-450(C21) on NaDodSO₄/polyacrylamide gel electrophoresis. After sucrose gradient sedimentation, mRNA encoding P-450(C21) was found in the 19S fraction. This fraction was reverse transcribed into double-stranded cDNA and inserted into the Pst I site of pBR322 by the dC·dG tailing procedure. Escherichia coli cells transformed with recombinant plasmids were screened with an in situ immunoassay using anti-P-450(C21) serum and ¹²⁵I-labeled staphylococcal protein A. Two colonies consistently bound anti P-450(C21) serum. They were identified as carrying the same plasmid by restriction mapping. This plasmid, PC21a, contains an insert of 520 base pairs. It hybridizes with mRNA encoding P-450(C21). The peptide encoded by the insert in pC21a is highly homologous to two peptides isolated from porcine P-450(C21) and shows limited homology to the P-450 induced by phenobarbital in rat liver. This clone may be useful in studying the molecular genetics of human congenital adrenal hyperplasia due to 21-hydroxylase deficiency.