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

J Biol Chem. 1993 Sep 15;268(26):19681-9.

Abstract

Enzymatically active human 17 alpha-hydroxylase cytochrome P450 (P450c17) 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 histidine residues at the carboxyl terminus, was placed in the pCWori+ expression vector. The modified human P450c17 was detected spectrophotometrically (400 nmol of P450c17/liter culture) and was found to be integrated into E. coli membranes. This previously inaccessible human P450 was 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 expected enzymatic activities of human P450c17 were reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase, giving turnover numbers of 8.0 nmol/min/nmol P450 for pregnenolone, 6.5 nmol/min/nmol P450 for progesterone, 0.06 nmol/min/nmol P450 for 17 alpha-hydroxypregnenolone, and no detectable activity for 17 alpha-hydroxyprogesterone. This system was utilized to study the molecular basis of a novel form of combined 17 alpha-hydroxylase, 17,20-lyase deficiency resulting from compound heterozygous mutations, a missense point mutation Tyr64(TAT)--> Ser (TCT), and an Ile112 duplication (ATCATC). Upon expression of these mutant proteins in E. coli, the Tyr64 mutant has 15% of the wild type 17 alpha-hydroxylase activity, whereas the Ile112 duplication shows no activity, results consistent with the observed clinical phenotype.

Publication types

  • Case Reports
  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adrenocorticotropic Hormone
  • Amino Acid Sequence
  • Androgens / blood
  • Animals
  • Base Sequence
  • Chorionic Gonadotropin
  • Cloning, Molecular
  • DNA / genetics
  • DNA / isolation & purification
  • Escherichia coli / genetics*
  • Gene Expression
  • Humans
  • Infant
  • Liver / enzymology
  • Male
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • NADPH-Ferrihemoprotein Reductase / metabolism
  • Plasmids
  • Point Mutation*
  • Rats
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Serine
  • Steroid 17-alpha-Hydroxylase / biosynthesis*
  • Steroid 17-alpha-Hydroxylase / genetics*
  • Steroid 17-alpha-Hydroxylase / metabolism
  • Steroids / blood
  • Steroids / urine
  • Testis / enzymology
  • Tyrosine

Substances

  • Androgens
  • Chorionic Gonadotropin
  • Recombinant Proteins
  • Steroids
  • Tyrosine
  • Serine
  • Adrenocorticotropic Hormone
  • DNA
  • Steroid 17-alpha-Hydroxylase
  • NADPH-Ferrihemoprotein Reductase