Engineering, expression, and purification of “soluble” human cytochrome P45017α and its functional characterization

Abstract

To engineer a "soluble" form of membrane-bound cytochrome P45017alpha (CYP17)--a key enzyme in steroid hormone biosynthesis--in the present work we have built a computer model of the tertiary structure of the hemeprotein, identified the surface hydrophobic amino acid residues, substituted these residues for more hydrophilic ones, and expressed and purified hydrophilized forms of CYP17. We have constructed and purified the following mutant forms of human CYP17: CYP17dH (CYP17 with deleted hydrophobic N-terminal sequence (Delta(23))) and CYP17mod (CYP17dH with substituted cluster of hydrophobic amino acid residues in the region of the FG-loop). Removal of the N-terminal sequence responsible for interaction with the membrane does not dramatically change the association of the protein with the membrane. However, CYP17mod containing hydrophilic FG-loop is mostly localized in the cytosolic fraction. Thus, in the present work we for the first time engineered a "soluble" form of the usually membrane-bound human CYP17 that is not bound to membrane. The expression degree of CYP17mod is approximately 900 nmol/liter of culture. The hemeprotein can be purified to apparent homogeneity without using detergents at any purification step. It is shown that replacement of hydrophobic amino acid residues in the FG-loop region does not change the metabolic profile during hydroxylation of steroids that is characteristic for wild type CYP17. Besides, the modification of the hemeprotein does not affect the affinity of CYP17 to steroid substrates. The engineered "soluble" form of human CYP17 is used as a subject for crystallization of the hemeprotein.

Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1

“…Recombinant human P450 17A1 (with a C-terminal His 4 tag) was expressed in E. coli and purified by metal-affinity chromatography using a protocol adapted from those previously reported (76,84,85). Briefly, an E. coli codon-optimized cDNA, corresponding to the amino acid sequence reported by DeVore and Scott (76), was purchased (Genewiz, South Plainfield, NJ) and inserted into the pCW-Ori(ϩ) expression vector.…”

Structures of cytochrome P450 17A1 with prostate cancer drugs abiraterone and TOK-001

“…Cells were grown, harvested, and disrupted as described 10,31 . After centrifugation (5000 × g), CYP17A1 was solubilized with either 4.8 mM Cymal-5 (for crystallography; Affymetrix, Santa Clara, CA) or 2% Emulgen 913 (for assays; Desert Biologicals, Phoenix, AZ), followed by ultracentrifugation (80,000 × g) for 60 minutes.…”

“…After centrifugation (5000 × g), CYP17A1 was solubilized with either 4.8 mM Cymal-5 (for crystallography; Affymetrix, Santa Clara, CA) or 2% Emulgen 913 (for assays; Desert Biologicals, Phoenix, AZ), followed by ultracentrifugation (80,000 × g) for 60 minutes. The lysate was loaded onto a NTA-agarose (Qiagen, Valencia, CA) column and purified as reported 31 . Eluted CYP17A1 fractions were pooled, diluted 5-fold with CM buffer (50mM Tris, pH 7.4, 20% glycerol, 100 mM glycine, 1 mM EDTA), and loaded on a HiTrap CM fast flow column (GE Healthcare, Uppsala, Sweden).…”

Substrate-modulated Cytochrome P450 17A1 and Cytochrome b5 Interactions Revealed by NMR

“…A form of CYP17A1 with a truncation of the N-terminal transmembrane helix and a C-terminal histidine tag was expressed and purified as described previously (21,33). Briefly, E. coli JM109 cells transformed with the CYP17A1 gene in a pCWori plasmid were grown in Terrific broth until log phase (ϳ0.6 optical density) and then induced using isopropyl 1-thio-␤-D-galactopyranoside and ALA ␥-aminolevulinic acid.…”