Purification and characterization of bovine steroid 21-hydroxylase (P450c21) efficiently expressed in Escherichia coli

Three-dimensional Structure of Steroid 21-Hydroxylase (Cytochrome P450 21A2) with Two Substrates Reveals Locations of Disease-associated Variants

“…Substrate Binding and Catalytic Activity of CYP21A2-Wildtype bovine CYP21A2 has been efficiently expressed in E. coli (16). At lower concentrations, the protein showed the expected catalytic activity, which suggests that the protein is stable and properly folded.…”

“…Preliminary experiments showed very similar rates of progesterone 21-hydroxylation by both a truncated P450 21A2 (16) and the C3B21RA mutant in 0.002% (w/v) Cymal 5 detergent and with 30 M L-␣-dilauroyl-sn-glycero-3-phosphocholine vesicles, a commonly used P450 reconstitution system. For the determination of steady-state kinetic parameters, incubations contained 2 pmol of P450 21A2 (either the truncated version (16) or the C3B21RA mutant), 60 pmol of E. coli recombinant NADPH-P450 reductase (17, 18), 2 g of L-␣-dilauroyl-sn-glycero-3-phosphocholine, and varying con- …”

“…The C3B21RA protein was expressed and purified as reported earlier (16). In brief, cells co-transformed with the plasmids CYP21A2pET17b and pGro12 were cultured overnight in LB broth containing 100 g/ml ampicillin and 50 g/ml kanamycin.…”

“…Catalytic Activity Assay-The procedure was modified from previous work (16). Preliminary experiments showed very similar rates of progesterone 21-hydroxylation by both a truncated P450 21A2 (16) and the C3B21RA mutant in 0.002% (w/v) Cymal 5 detergent and with 30 M L-␣-dilauroyl-sn-glycero-3-phosphocholine vesicles, a commonly used P450 reconstitution system.…”

The cytochrome P450 gene family CYP157 does not contain EXXR in the K‐helix reducing the absolute conserved P450 residues to a single cysteine

“…As previous heterologous expression of CYP157C1 in E. coli produced extremely low levels of protein (<10 nmol CYP/L culture) preventing biochemical analysis of this protein [11], we established an efficient system for expression by coexpressing it with the molecular chaperones GroES and GroEL that have been shown to enhance the production of active and correctly folded human CYPs [15,16]. In this co-expression system, GroES and GroEL coding regions are under the control of the AraB promoter allowing them to be induced by the addition of arabinose.…”