Heterologous expression of the spiny dogfish shark (Squalus acanthias) cytochrome P450c17 (17 alpha-hydroxylase) in Escherichia coli.
Cytochrome P450c17 is a key steroidogenic enzyme for the production of sex steroids in gonadal tissue and for cortisol production in adrenal tissue. This protein possesses two enzymatic activities. The 17-alpha-hydroxylase activity introduces a hydroxyl group into the steroid nucleus. The resultant 17-alpha-hydroxylated, C(21) pregnenes can be converted to a C(19) androgen by the C(17,20)-lyase activity. The cDNA encoding the spiny dogfish shark (Squalus acanthias) testicular form of cytochrome P450c17 was used to direct heterologous expression in Escherichia coli. The wild-type P450c17 protein was not conducive to expression in E. coli using either a pET21 or a pCwori+ vector. However, modification of the amino terminus permitted overexpression of inactive shark protein with the pFT21 vector. This protein was fused with a hexahistidinyl peptide to facilitate purification by column chromatography using a Ni(2+)-chelated resin. Transformed bacteria yielded an average of 1.8 mg of purified, concentrated P450c17 protein per 100-ml culture. This modified protein was used to raise antisera in rabbits and the resultant antisera was used at a working titer of 1:10,000 for Western blot analysis. Culture conditions that result in the accumulation of bioactive recombinant bovine P450c17 failed to demonstrate expression of either the native or the modified form of shark P450c17 using the pCWori+ and pET21 vectors. These results suggest that the amino terminus of the native shark P450c17 was not conducive to synthesis in E. coli. However, modifications of the amino terminus permitted synthesis of an inactive protein that was protected from degradation within inclusion bodies.
Trant
J M
JM
Department of Zoology & Physiology, Louisiana State University, Baton Rouge 70803, USA.
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D004284:Dogfish / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D005818:Genetic Engineering; D008297:Male; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D013254:Steroid 17-alpha-Hydroxylase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D013737:Testis / Q000201:enzymology / Q000648:ultrastructure
D013254:Steroid 17-alpha-Hydroxylase