EXPRESSION AND PURIFICATION OF FUNCTIONAL HUMAN 17-ALPHA-HYDROXYLASE 17,20-LYASE (P450C17) IN ESCHERICHIA-COLI - USE OF THIS SYSTEM FOR STUDY OF A NOVEL FORM OF COMBINED 17-ALPHA-HYDROXYLASE/17,20-LYASE DEFICIENCY/

Enzymatically active human 17alpha-hydroxylase cytochrome P450 (P45Oc1 7) has been expressed in and purified from Escherichia coli. The cDNA containing modifications within the amino-terminal eight codons which are favorable for expression in E. coli, as well as codons for 4 histi dine residues at the carboxyl terminus, was placed in the pCWori+ expr ession vector. The modified human P45Oc17 was detected spectrophotomet rically (400 nmol of P45Oc17/liter culture) and was found to be integr ated into E. coli membranes. This previously inaccessible human P450 w as purified to electrophoretic homogeneity (10.7 nmol of P450/mg) from solubilized bacterial membranes using two sequential chromatographic steps, nickel nitrilotriacetate followed by hydroxylapatite. The expec ted enzymatic activities of human P45Oc17 were reconstituted by additi on of purified rat liver NADPH-cytochrome P450 reductase, giving turno ver numbers of 8.0 nmol/min/nmol P450 for pregnenolone, 6.5 nmol/min/n mol P450 for progesterone, 0.06 nmol/min/nmol P450 for 17alpha-hydroxy pregnenolone, and no detectable activity for 17alpha-hydroxyprogestero ne. This system was utilized to study the molecular basis of a novel f orm of combined 17alpha-hydroxylase, 17,20-lyase deficiency resulting from compound heterozygous mutations, a missense point mutation Tyr64( TAT) --> Ser (TCT), and an Ilel112 duplication (ATCATC). Upon expressi on of these mutant proteins in E. coli, the Tyr64 mutant has 15% of th e wild type 17alpha-hydroxylase activity, whereas the Ile112 duplicati on shows no activity, results consistent with the observed clinical ph enotype.