Expression and purification of human cholesterol 7~ hydroxylase in Escherichia coli

Cholesterol 'la-hydroxylase (P450c7) is the first and rate-limiting enzyme in bile acid biosynthesis and is the product of a cytochrome P450 gene, CYP7. We have previously reported the cloning of a full-length human cholesterol 'la-hydroxylase cDNA (Karam, W. G., and J. Y. L. Chiang. 1992. Biochem. Biophys. Res. Commun. 185: 588-595). Using this clone in a polymerase chain reaction, we have generated a cDNA (H7a1.5) in which the codons for the N-terminal 24 amino acid residues were deleted. The translational product of this cDNA would be a truncated protein, P45Oc7(A 2-24) with a hydrophilic NH2terminal sequence, Met-Ala-Arg-Arg-Arg-Gln ... This cDNA was cloned into the expression vector pJL and the construct pJL/H7a1.5 was transformed into E. coli strain TOPP3. We have also ligated a truncated rat cholesterol 7a-hydroxylase cDNA obtained previously (Li, Y. C., and J. Y. L. Chiang. 1991. J. Biol. Chem. 266: 19186-19191) into the pJL vector and have transformed this construct (pJL/R7a1.5) into E. coli strain MV1304. Both of these systems expressed functional cholesterol 7a-hydroxylase in E. coli. A fivefold improvement in the expression of rat enzyme over the previous expression system was obtained. About 70-80% of the truncated human P450 in the clear lysate was localized in the cytosol. The truncated human and rat P45Oc7(A 2-24) were purified to homogeneity. Reconstitution of cholesterol 7a-hydroxylase activity using purified rat or human P45Oc7(A 2-24) showed a similar K , of 6 and 7 PM for cholesterol, a V,, of 0.13 and 0.14 nmol/min, and a turnover number of 1.3 and 1.5 per min, respectively. Immunoblotting experiment revealed that a polyclonal antibody raised against rat microsomal cholesterol 'la-hydroxylase recognized both rat and human P45Oc7(A 2-24). I This expression system provides a method for isolation of a large quantity of purified and catalytically active cholesterol 7a-hydroxylase for the study of structure and function of this important enzyme in bile acid synthesis and cholesterol homeostasis.Karam, W. G., and J. Y. L. Chiang. Expression and purification of human cholesterol 7a hydroxylase in Escherichia coli. J. Lipid Res. 1994. 35: 1222-1231. Supplementary key words bile acid synthesis cytochrome P450 cDNA Cholesterol 7cy-hydroxylase (P450c7) (EC.1.14.13.17), the product of the CYP7 gene (l), is the first and ratelimiting enzyme in the conversion of cholesterol to bile acids in the liver (2, 3). This enzyme activity is feedbackregulated by hydrophobic bile acids returning to the liver via the enterohepatic circulation of the bile (2) and is stimulated by treatment with cholestyramine, a bile acid sequestrant, and by feeding a high cholesterol diet to rats (4). Recent breakthroughs in the purification of rat cholesterol 7a-hydroxylase (5, 6) and cloning of cDNAs (4, 7, 8) and the gene encoding rat cholesterol 7a-hydroxylase (9, 10) have contributed to the understanding of molecular mechanism of regulation of bile acid synthesis by this rate-limiting enzyme. It has been demonstrated that the changes in microsomal cholesterol 7a-hydroxylase activity and enzyme levels parallel the steady-state mRNA levels and the rate of gene transcription in rat livers (4, 5, 7, 8, 11). However, regulation of cholesterol 7a-hydroxylase in the human liver has not been studied as extensively as in the rat liver. This is partly due to the lack of suitable human liver tissues and the expression of an extremely low level of cholesterol 7cy-hydroxylase activity in human liver, which prevented the purification and characterization of human cholesterol 7cy-hydroxylase. Two laboratories have reported partial purification of the human cholesterol 7a-hydroxylase from human liver; however, kinetic characteristics of the human enzyme have not been studied (12, 13). Full-length cDNAs encoding human cholesterol 7a-hydroxylase have been cloned recently by screening human liver cDNA libraries using rat cDNAs as hybridization probes (14, 15). Recent advances in the expression of membrane-bound proteins such as P450 isozymes in heterologous system, E. coli, have provided a unique method for the production and isolation of human enzymes (16-18). Abbreviations: PCR, polymerase chain reaction; LB broth, Luria Bertani broth; IPTG, isopropyl-6-D-thiogalactopyranoside; HPLC, high performance liquid chromatography; DTT, dithiothreitol; EDTA, ethylenediamine tetraacetic acid; PMSF, phenylmethyl sulfonylfluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; &ALA, delta-aminolevulinic acid; OAS-4B, octylamino-Sepharose B; CHAPS, 3-[(3-cholamidopropyI)dimethyl-ammoniodimethylammonio]-lpropanesulfonate. 'To whom correspondence should be addressed. 1222 Journal of Lipid Research Volume 35, 1994 by gest, on S etem er 0, 2017 w w w .j.org D ow nladed fom In this communication, we report the development of an E. coli expression system for a human cholesterol 7ahydroxylase based on our previous strategy of expressing a truncated, soluble, and catalytically active rat cholesterol 7a-hydroxylase in E. coli (16). The bacteriaexpressed human cholesterol 7a-hydroxylase was purified and its kinetic and immunochemical properties were characterized. EXPERIMENTAL PROCEDURES