TY - JOUR
T1 - Expression of functional bovine cholesterol side chain cleavage cytochrome P450 (P450scc) in Escherichia coli
AU - Wada, Akira
AU - Mathew, Porunelloor A.
AU - Barnes, Henry J.
AU - Sanders, Donita
AU - Estabrook, Ronald W.
AU - Waterman, Michael R.
N1 - Funding Information:
This research was supported in part by USPHS Grants GM37942 and GM16488 and Grant I-624 from The Robert A. Welch Foundation. A.W. is supported in part by a grant from the Japanese Heart Foundation. H.J.B. is supported in part by NIH Training Grant #5-T32-GM08203-04. The authors appreciate the assistance of Dr. Michael P. Arlotto in carrying out HPLC analysis of steroid products and the generosity of Dr. Egon Amann in providing the Trc99A expression vector.
PY - 1991/11/1
Y1 - 1991/11/1
N2 - Escherichia coli expression vectors containing the trc promoter and the complete DNA sequence of either the precursor or the mature form of bovine adrenocortical cholesterol side chain cleavage cytochrome P450 (P450scc) were transformed into E. coli strain JM109 and transcription induced with isopropyl-β-d-thiogalactopyranoside (IPTG). Immunoreactive cytochrome P450scc was produced using the plasmid containing the mature P450scc sequence but not with the plasmid containing the sequence of the precursor form of P450scc, even though P450scc RNA was detectable in both cases. The mature form of P450scc was detected spectrophotometrically in a reduced CO-difference spectrum in E. coli (40-60 nmol/liter culture). Cholesterol and hydroxylated derivatives (22-hydroxycholesterol and 25-hydroxycholesterol) produce a type 1 substrate-binding spectrum in IPTG-induced, transformed E. coli. The P450scc was found to be associated with the E. coli membrane fraction and the enzymatic activity of side chain cleavage of 25-hydroxycholesterol was reconstituted using solubilized membranes, in the presence of purified bovine adrenocortical adrenodoxin and NADPH-adrenodoxin reductase (turnover number; 15.4 nmol/min/nmol P450). This bacterial expression system provides functional P450scc, in the absence of other forms of P450, which can be used for evaluation of enzymatic and spectral properties of this mitochondrial P450 by site-directed mutagenesis.
AB - Escherichia coli expression vectors containing the trc promoter and the complete DNA sequence of either the precursor or the mature form of bovine adrenocortical cholesterol side chain cleavage cytochrome P450 (P450scc) were transformed into E. coli strain JM109 and transcription induced with isopropyl-β-d-thiogalactopyranoside (IPTG). Immunoreactive cytochrome P450scc was produced using the plasmid containing the mature P450scc sequence but not with the plasmid containing the sequence of the precursor form of P450scc, even though P450scc RNA was detectable in both cases. The mature form of P450scc was detected spectrophotometrically in a reduced CO-difference spectrum in E. coli (40-60 nmol/liter culture). Cholesterol and hydroxylated derivatives (22-hydroxycholesterol and 25-hydroxycholesterol) produce a type 1 substrate-binding spectrum in IPTG-induced, transformed E. coli. The P450scc was found to be associated with the E. coli membrane fraction and the enzymatic activity of side chain cleavage of 25-hydroxycholesterol was reconstituted using solubilized membranes, in the presence of purified bovine adrenocortical adrenodoxin and NADPH-adrenodoxin reductase (turnover number; 15.4 nmol/min/nmol P450). This bacterial expression system provides functional P450scc, in the absence of other forms of P450, which can be used for evaluation of enzymatic and spectral properties of this mitochondrial P450 by site-directed mutagenesis.
UR - http://www.scopus.com/inward/record.url?scp=0026094652&partnerID=8YFLogxK
U2 - 10.1016/0003-9861(91)90554-V
DO - 10.1016/0003-9861(91)90554-V
M3 - Article
C2 - 1929405
AN - SCOPUS:0026094652
SN - 0003-9861
VL - 290
SP - 376
EP - 380
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 2
ER -