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/
T. Imai et al., 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/, The Journal of biological chemistry, 268(26), 1993, pp. 19681-19689
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.