PURIFICATION AND CHARACTERIZATION OF RECOMBINANT-EXPRESSED CYTOCHROME-P450-2C3 FROM ESCHERICHIA-COLI - 2C3 ENCODES THE 6-BETA-HYDROXYLASE DEFICIENT FORM OF P450-3B

被引:60
作者
RICHARDSON, TH
HSU, MH
KRONBACH, T
BARNES, HJ
CHAN, G
WATERMAN, MR
KEMPER, B
JOHNSON, EF
机构
[1] SCRIPPS RES INST, DEPT MOLEC & EXPTL MED, DIV BIOCHEM NX4, LA JOLLA, CA 92037 USA
[2] UNIV TEXAS, HLTH SCI CTR, SW MED SCH, DALLAS, TX 75235 USA
[3] UNIV ILLINOIS, URBANA, IL 61801 USA
关键词
D O I
10.1006/abbi.1993.1069
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Rabbit cytochrome P450 2C3 was expressed from its cDNA in Escherichia coli as a chimeric enzyme in which a portion of the N-terminal membrane anchor sequence of 2C3 was replaced with a modified sequence derived from P450 17α. The nucleotide sequence encoding the N-terminus of P450 17α was modified previously to achieve a high level of expression of P450 17α in E. coli by altering the first eight codons of P450 17α to reflect second codon preferences for high expression and to minimize the potential for the formation of a stable secondary structure of the corresponding RNA transcript. The modified P450 2C3 was expressed at >400 nmol/liter of culture. P450 2C3 was isolated to apparent electrophoretic homogeneity and a specific content >14 nmol P450/mg protein. When reconstituted with P450 reductase and dilauroyl-L-α-lecithin, the purified E. coli-expressed P450 2C3 catalyzed 16α, but not 6β-hydroxylation of progesterone. Expression of unmodified 2C3 from its cDNA in COS-1 cells confirmed the absence of detectable 6β-hydroxylase activity. In addition, the enzyme expressed in E. coli is activated by the allosteric effector 5β-pregnane-3β,20α-diol, with a resultant V(max) = 10 min-1 and K(m) = 20 μM and is not inhibited by 16α-methylprogesterone. These results indicate that the 2C3 cDNA encodes an enzymatic form characteristic of IIIvo/J and B/J inbred rabbits rather than a second enzymatic form expressed in most outbred and some inbred strains that catalyzes both high efficiency 16α- and 6β-hydroxylation of progesterone. Our results have identified the enzyme variant encoded by the 2C3 cDNA and have demonstrated the utility of E. coli for the expression of recombinant P450 enzymes. © 1993 Academic Press, Inc.
引用
收藏
页码:510 / 516
页数:7
相关论文
共 37 条
  • [21] LI YC, 1991, J BIOL CHEM, V266, P19186
  • [22] INFLUENCE OF THE CODON FOLLOWING THE AUG INITIATION CODON ON THE EXPRESSION OF A MODIFIED LACZ GENE IN ESCHERICHIA-COLI
    LOOMAN, AC
    BODLAENDER, J
    COMSTOCK, LJ
    EATON, D
    JHURANI, P
    DEBOER, HA
    VANKNIPPENBERG, PH
    [J]. EMBO JOURNAL, 1987, 6 (08) : 2489 - 2492
  • [23] MICROSOMAL NICOTINE METABOLISM - A COMPARISON OF RELATIVE ACTIVITIES OF 6 PURIFIED RABBIT CYTOCHROME-P-450 ISOZYMES
    MCCOY, GD
    DEMARCO, GJ
    KOOP, DR
    [J]. BIOCHEMICAL PHARMACOLOGY, 1989, 38 (07) : 1185 - 1188
  • [24] METABOLIC PROCESSING OF 2-ACETYLAMINOFLUORENE BY MICROSOMES AND 6 HIGHLY PURIFIED CYTOCHROME-P-450 FORMS FROM RABBIT LIVER
    MCMANUS, ME
    MINCHIN, RF
    SANDERSON, N
    SCHWARTZ, D
    JOHNSON, EF
    THORGEIRSSON, SS
    [J]. CARCINOGENESIS, 1984, 5 (12) : 1717 - 1723
  • [25] THE P450 SUPERFAMILY - UPDATE ON NEW SEQUENCES, GENE-MAPPING, AND RECOMMENDED NOMENCLATURE
    NEBERT, DW
    NELSON, DR
    COON, MJ
    ESTABROOK, RW
    FEYEREISEN, R
    FUJIIKURIYAMA, Y
    GONZALEZ, FJ
    GUENGERICH, FP
    GUNSALUS, IC
    JOHNSON, EF
    LOPER, JC
    SATO, R
    WATERMAN, MR
    WAXMAN, DJ
    [J]. DNA AND CELL BIOLOGY, 1991, 10 (01) : 1 - 14
  • [26] NELSON DR, 1988, J BIOL CHEM, V263, P6038
  • [27] NORMAN RL, 1978, J BIOL CHEM, V253, P8640
  • [28] OMURA T, 1964, J BIOL CHEM, V239, P2379
  • [29] OZOLS J, 1985, J BIOL CHEM, V260, P5427
  • [30] PORTER TD, 1991, METHOD ENZYMOL, V206, P108