Expression of human 21-hydroxylase (P450c21) in bacterial and mammalian cells: a system to characterize normal and mutant enzymes.
Cytochrome P450c21 (steroid 21-hydroxylase) is a key enzyme in the synthesis of cortisol, whose deficiency is the cause of a common genetic disease, congenital adrenal hyperplasia. We have expressed P450c21 (steroid 21-hydroxylase) in E. coli and mammalian cells. In E. coli, P450c21 cDNA was cloned into a T7 expression vector to produce a large amount of P450c21 fusion protein, which enabled antiserum production. In mammalian cells, a plasmid containing full-length P450c21 cDNA (phc21) was constructed and transfected into COS-1 cells to produce active P450c21, which was detected by immunoblotting and 21-hydroxylase activity assay. This system was used to assay mutations involved in the disease. Ile172 of phc21 corresponding to the site of mutation in some cases of the disease was mutagenized to become Asn, Leu, His, or Gln. Mutant as well as normal P450c21 was produced when their cDNAs were transfected into COS-1 cells. The mutant proteins, however, had greatly reduced 21-hydroxylase activities. Therefore, missense mutation at Ile172 resulted in inactivation of the enzyme, but not in repression of enzyme synthesis. The Leu for Ile substitution at amino acid 172 did not result in partial restoration of enzymatic activity, indicating that hydrophobicity at this residue may not play a role in its function.
Hu
M C
MC
Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China.
Chung
B C
BC
D000312:Adrenal Hyperplasia, Congenital; D000595:Amino Acid Sequence; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D015870:Gene Expression; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D013255:Steroid 21-Hydroxylase / Q000235:genetics* / Q000302:isolation & purification; D014162:Transfection
D003577:Cytochrome P-450 Enzyme System; D013255:Steroid 21-Hydroxylase