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.
Karam
W G
WG
Department of Biochemistry and Molecular Pathology, Northeastern Ohio Universities College of Medicine, Rootstown 44272.
Chiang
J Y
JY
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