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A general procedure for the solubilization of cytochrome P450 (P450) from bacterial membranes specifically for a human P450 expressed heterologously in the host Escherichia coli is described. The example involves the use of a P450 (3A4) with a C-terminal oligohistidine tag and includes sequential DEAE and metal affinity chromatography.
A large number of microsomal P450s have been expressed in Escherichia coli in quantities sufficient for structure/function analysis. However, only one mitochondrial P450 has been successfully overexpressed, that being cholesterol side chain cleavage cytochrome P450 (P450scc). We report here overexpression, purification, and characterization of a second mitochondrial P450, human sterol C-27 hydroxylase (P450c27). The conditions used for expression are very similar to those applied for P450scc, although a quite different purification protocol was necessary to achieve highly purified P450c27. The catalytic properties of purified recombinant human P450c27 resemble those of purified, endogenous rat and rabbit P450c27, regarding both specificity and turnover numbers. Like endogenous P450c27 from rat and rabbit liver, human recombinant P450c27 is only functional in the presence of adrenodoxin and adrenodoxin reductase and shows no activity in the presence of the microsomal P450 reductase. We conclude that P450c27 is most likely not the 1alpha-hydroxylase of 25-hydroxyvitamin D3, contrary to a previous suggestion (Axen, E., Postlind, H., Sjöberg, H., and Wikvall, K. (1994) Proc. Natl. Acad. Sci. USA 91, 10014-10018) because this activity of P450c27 (28 pmol/min/nmol P450) seems far too low to be physiologically relevant. This activity is 10(3) times lower than the 27-hydroxylase activity toward 5beta-cholestane-3alpha,7alpha,12alpha-triol and 40 times lower than the 27-hydroxylation of cholesterol by this enzyme. The development of this expression system and purification procedure creates the potential for structure/function analysis of P450c27, including possible crystallization of this important enzyme.
A plasmid (pCW) was modified to code for a fusion protein consisting of the complete sequence of human cytochrome P450 (P450) 1A1 (with only the second amino acid changed) in the N-terminal portion connected by a Ser-Thr linker to the portion of rat NADPH-P450 reductase beginning at amino acid 57. This plasmid was used to express the fusion protein in Escherichia coli DH5alpha cells and the protein was purified from detergent-solubilized bacterial membranes using DEAE and 2',5'-ADP agarose chromatography. The purified fusion protein catalyzed benzo[a]pyrene 3-hydroxylation, 7-ethoxyresorufin O-deethylation, and zoxazolamine 6-hydroxylation. Catalytic activity was not increased in the presence of added NADPH-P450 reductase, cytochrome b5, or phospholipid. The fusion protein could also transfer electrons to cytochromes c and b5 but not P450 lA2. The same oxidation products of benzo[a]pyrene were formed with the purified fusion protein and the fusion protein functioning in bacterial cells. The catalytic activity of the human P450 1A1 fusion protein toward several substrates is markedly less than that of a similar fusion protein constructed with rat P450 1A1, in line with the reported differences in catalytic activities of the rat and human P450 1A1 enzymes. The purified fusion protein also oxidized (+)- and (-)-benzo[a]pyrene 7,8-dihydrodiols and eight aryl and heterocyclic amines to genotoxic products, in the absence of added NADPH-P450 reductase. The demonstration of catalytic activities of the human fusion protein within bacterial cells suggests the prospect of utilizing such cellular systems for production of human P450 metabolites.
Rabbit cytochrome P450 2C3 was expressed from its cDNA in Escherichia coli as a chimeric enzyme in which a portion of the N-terminal membrane anchor sequence of 2C3 was replaced with a modified sequence derived from P450 17 alpha. The nucleotide sequence encoding the N-terminus of P450 17 alpha was modified previously to achieve a high level of expression of P450 17 alpha in E. coli by altering the first eight codons of P450 17 alpha to reflect second codon preferences for high expression and to minimize the potential for the formation of a stable secondary structure of the corresponding RNA transcript. The modified P450 2C3 was expressed at > 400 nmol/liter of culture. P450 2C3 was isolated to apparent electrophoretic homogeneity and a specific content > 14 nmol P450/mg protein. When reconstituted with P450 reductase and dilauroyl-L-alpha-lecithin, the purified E. coli-expressed P450 2C3 catalyzed 16 alpha, but not 6 beta-hydroxylation of progesterone. Expression of unmodified 2C3 from its cDNA in COS-1 cells confirmed the absence of detectable 6 beta-hydroxylase activity. In addition, the enzyme expressed in E. coli is activated by the allosteric effector 5 beta-pregnane-3 beta,20 alpha-diol, with a resultant Vmax = 10 min-1 and Km = 20 microM and is not inhibited by 16 alpha-methylprogesterone. These results indicate that the 2C3 cDNA encodes an enzymatic form characteristic of IIIvo/J and B/J inbred rabbits rather than a second enzymatic form expressed in most outbred and some inbred strains that catalyzes both high efficiency 16 alpha- and 6 beta-hydroxylation of progesterone. Our results have identified the enzyme variant encoded by the 2C3 cDNA and have demonstrated the utility of E. coli for the expression of recombinant P450 enzymes.
The high molecular weight folate binding protein of rat liver cytosol has been purified to apparent homogeneity. Purification was achieved by using a combination of gel filtration, O-(diethylaminoethyl)cellulose chromatography, and affinity chromatography. This folate binding protein was initially identified during purification by an in vivo labeling procedure involving intraperitoneal injection of [3H]folic acid prior to sacrifice and subsequently by its ability to bind naturally reduced [3H]folate polyglutamates in vitro. A molecular weight of 210 000 was estimated by gel chromatography. This is distinct from the trifunctional formyl-methenyl-methylene synthetase of rat liver which has a molecular weight of 225 000. Sodium dodecyl sulfate electrophoresis revealed a single band with a molecular weight of about 100 000 which suggests the native protein is composed of two identical subunits. The partially purified protein contains bound tetrahydropteroylpentaglutamate.
We have purified and characterized an oligopeptide binding protein released from the periplasm of Escherichia coli W by mild osmotic shock. The purified protein was greater than 97% homogeneous as determined by either sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 60,000) or isoelectric focusing (pI = 5.95). The binding protein has a Stokes radius of 30 A and a sedimentation coefficient (s(0)20,w) of 4.6 S. Based on these hydrodynamic studies, the native protein has a molecular weight of 56,000. The tripeptide, Ala-Phe-[3H]Gly, which is transported via the shock-sensitive sensitive oligopeptide permease, binds to the purified protein in dilute solution with a Kd of 0.1 microM and a stoichiometry of approximately 1 to 1. Results from this study support the hypothesis that this periplasmic oligopeptide binding protein functions in the initial recognition of peptide substrates for the oligopeptide permease system.