Expression and purification of human cholesterol 7 alpha-hydroxylase in Escherichia coli.
Cholesterol 7 alpha-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 7 alpha-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 (H7 alpha 1.5) 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, P450c7(delta 2-24) with a hydrophilic NH2-terminal sequence, Met-Ala-Arg-Arg-Arg-Gln... This cDNA was cloned into the expression vector pJL and the construct pJL/H7 alpha 1.5 was transformed into E. coli strain TOPP3. We have also ligated a truncated rat cholesterol 7 alpha-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/R7 alpha 1.5) into E. coli strain MV1304. Both of these systems expressed functional cholesterol 7 alpha-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 P450c7(delta 2-24) were purified to homogeneity. Reconstitution of cholesterol 7 alpha-hydroxylase activity using purified rat or human P450c7(delta 2-24) showed a similar Km of 6 and 7 microM for cholesterol, a Vmax 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 7 alpha-hydroxylase recognized both rat and human P450c7(delta 2-24). This expression system provides a method for isolation of a large quantity of purified and catalytically active cholesterol 7 alpha-hydroxylase for the study of structure and function of this important enzyme in bile acid synthesis and cholesterol homeostasis.
Department of Biochemistry and Molecular Pathology, Northeastern Ohio Universities College of Medicine, Rootstown 44272.
W G
WG
Karam
J Y
JY
Chiang
D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002790:Cholesterol 7-alpha-Hydroxylase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000302:isolation & purification; D004926:Escherichia coli; D006801:Humans; D007120:Immunochemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D012995:Solubility; D013347:Subcellular Fractions / Q000201:enzymology
D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D002790:Cholesterol 7-alpha-Hydroxylase