EXPRESSION AND PURIFICATION OF HUMAN CHOLESTEROL 7-ALPHA-HYDROXYLASE IN ESCHERICHIA-COLI

Cholesterol 7 alpha-hydroxylase (P450c7) is the first and rate-limitin g enzyme in bile acid biosynthesis and is the product of a cytochrome P450 gene, CYP7. We have previously reported the cloning of a full-len gth 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 res idues 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 exp ression vector pJL and the construct pJL/H7 alpha 1.5 was transformed into E. coli strain TOPP3. We have also ligated a truncated rat choles terol 7 alpha-hydroxylase cDNA obtained previously (Li, Y. C., and J. Y. L. Chiang. 1991. J. Biol. Chem. 266: 19186-19191) into the pJL vect or 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 express ion of rat enzyme over the previous expression system was obtained. Ab out 70-80 % of the truncated human P450 in the clear lysate was locali zed in the cytosol. The truncated human and rat P450c7(Delta 2-24) wer e purified to homogeneity. Reconstitution of cholesterol 7 alpha-hydro xylase activity using purified rat or human P450c7(Delta 2-24) showed a similar K-m of 6 and 7 mu M for cholesterol, a V(m)a(x) of 0.13 and 0.14 nmol/min, and a turnover number of 1.3 and 1.5 per min, respectiv ely. Immunoblotting experiment revealed that a polyclonal antibody rai sed 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 catalytica lly active cholesterol 7 alpha-hydroxylase for the study of structure and function of this important enzyme in bile acid synthesis and chole sterol homeostasis.