The expression of a catalytically active cholesterol 7 alpha-hydroxylase cytochrome P450 in Escherichia coli.

Title

The expression of a catalytically active cholesterol 7 alpha-hydroxylase cytochrome P450 in Escherichia coli.

Creator

Li Y C; Chiang J Y

Publisher

The Journal of biological chemistry

Date

1991
1991-10

Description

We have recently cloned a full-length cDNA encoding the rat hepatic cholesterol 7 alpha-hydroxylase cytochrome P450 (P450c7) (Li, Y. C., Wang, D. P., and Chiang, J. Y. L. (1990) J. Biol. Chem. 265, 12012-12019), which catalyzes the rate-limiting reaction of bile acid synthesis in the liver. By using the polymerase chain reaction, we have designed two P450c7 cDNAs. One has the second Met codon deleted and the third Thr codon replaced with an Ala. The other lacks codons for the NH2-terminal hydrophobic sequence of amino acids 2-24 (P450c7 delta 2-24). The cDNAs were separately cloned into the expression vector pKK233-2 and transformed into Escherichia coli. After induction with isopropyl-beta-D-thiogalactopyranoside, bacteria harboring recombinant plasmids expressed a polypeptide which reacted with the antibody against cholesterol 7 alpha-hydroxylase in immunoblots. The slightly modified full-length enzyme was expressed to 0.2% of the total bacterial lysate and was located in the membrane fraction, whereas P450c7 delta 2-24 was expressed at a 10-fold higher level (2%), of which 85% was in the cytosol and the remaining associated with the membranes. We have purified P450c7 delta 2-24 which showed a typical reduced-CO difference spectrum of cytochrome P450 and reconstituted cholesterol 7 alpha-hydroxylase activity in the presence of NADPH-cytochrome P450 reductase. P450c7 delta 2-24 has a similar Km for cholesterol (24.6 microM) but a lower Vmax (0.10 nmol/min) and a lower turnover number (1.93 min-1) as compared with the enzyme isolated from rat liver microsomes. The purified P450c7 delta 2-24 has an unique hydrophilic NH2 terminus and contains monomers and dimers in equal amounts. This is the first report demonstrating that a genetically engineered cytochrome P450 enzyme lacking a typical NH2-terminal hydrophobic sequence is mainly cytosolic and catalytically active.

Subject

Gene Expression; Amino Acid Sequence; Base Sequence; Polymerase Chain Reaction; Liver/enzymology; Catalysis; Molecular Sequence Data; Substrate Specificity; Cholesterol/metabolism; Cholesterol 7-alpha-Hydroxylase/*genetics; DNA/genetics; Codon; Escherichia coli/*enzymology; Plasmids; Chromatography; Blotting; Western; Electrophoresis; Polyacrylamide Gel; Liquid; Microsomes

Rights

Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).

Pages

19186–19191

Issue

29

Volume

266

Citation

Li Y C; Chiang J Y, “The expression of a catalytically active cholesterol 7 alpha-hydroxylase cytochrome P450 in Escherichia coli.,” NEOMED Bibliography Database, accessed May 11, 2021, https://neomed.omeka.net/items/show/5515.

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