Expression in Escherichia coli of Functional Cytochrome P450c17 Lacking Its Hydrophobic Amino-Terminal Signal Anchor

doi:10.1006/abbi.1993.1349

Archives of Biochemistry and Biophysics

Volume 304, Issue 1, July 1993, Pages 272-278

https://doi.org/10.1006/abbi.1993.1349 Get rights and content

Abstract

A truncated form of bovine microsomal 17α-hydroxylase cytochrome P450 (P450_c17) lacking its amino-terminal hydrophobic signal anchor sequence (Δ2-17) was expressed in Escherichia coli to give more than 600 nmol P450 per liter using XL1-blue as the host. The expression level of the truncated P450_c17 showed variation among different host strains. When intact E. coli cells were used for analysis, the truncated P450_c17 showed the typical cytochrome P450 CO-difference spectrum and type I substrate binding spectra for pregnenolone, 17α-hydroxypregnenolone, progesterone, and 17α-hydroxyprogesterone. The apparent K₈ values for these steroids are comparable to those obtained under the same conditions with wild type P450_c17. The truncated P450_c17 is primarily associated with the membrane fraction from E. coli and remained with this fraction after treatment with 0.1 M Na₂CO₃. The 17α-hydroxylase activity of the truncated form was observed using intact E. coli. Using isolated membrane fractions, the truncated P450_c17 also showed 17,20-lyase activity upon reconstitution with rat liver microsomal NADPH-cytochrome P450 reductase. These results indicate that P450_c17 can be associated with E. coli membranes not only via the amino-terminal hydrophobic region as expected for the wild type, nontruncated form, but also through other sequences within the polypeptide chain and that integration of the amino- terminal region into the membrane is not essential for the folding pathway leading to functional P450_c17 in E. coli. This is in contrast to mammalian cells where the same truncated P450_c17 is found to be inactive and the folding pathway leading to functional P450_c17 appears to require a signal anchor sequence.

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Regular ArticleExpression in Escherichia coli of Functional Cytochrome P450c17 Lacking Its Hydrophobic Amino-Terminal Signal Anchor

Abstract

Regular Article
Expression in Escherichia coli of Functional Cytochrome P450_c17 Lacking Its Hydrophobic Amino-Terminal Signal Anchor