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Purification of cytochromes P450: products of bacterial recombinant expression systems.
Guengerich FP, Martin MV
(2006) Methods Mol Biol 320: 31-7
MeSH Terms: Chromatography, Affinity, Chromatography, DEAE-Cellulose, Cytochrome P-450 Enzyme System, Escherichia coli, Recombinant Proteins
Show Abstract · Added March 5, 2014
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.
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5 MeSH Terms
Purification of cytochromes P450: products of bacterial recombinant expression systems.
Guengerich FP, Hosea NA, Martin MV
(1998) Methods Mol Biol 107: 77-83
MeSH Terms: Bacteria, Chromatography, Affinity, Chromatography, DEAE-Cellulose, Cloning, Molecular, Cytochrome P-450 Enzyme System, Durapatite, Genetic Vectors
Added March 5, 2014
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7 MeSH Terms
Purification of cytochromes P450. Rat and human hepatic forms.
Guengerich FP, Martin MV
(1998) Methods Mol Biol 107: 35-53
MeSH Terms: Animals, Chromatography, DEAE-Cellulose, Cytochrome P-450 Enzyme System, Durapatite, Humans, Liver, Microsomes, Liver, Rats
Added March 5, 2014
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8 MeSH Terms
Expression, purification, and enzymatic properties of recombinant human cytochrome P450c27 (CYP27).
Pikuleva IA, Björkhem I, Waterman MR
(1997) Arch Biochem Biophys 343: 123-30
MeSH Terms: Amino Acid Sequence, Animals, Cholestanetriol 26-Monooxygenase, Cholestanols, Chromatography, DEAE-Cellulose, Chromatography, High Pressure Liquid, Cloning, Molecular, Cytochrome P-450 Enzyme System, Electrophoresis, Polyacrylamide Gel, Escherichia coli, Humans, Hydroxylation, Mutation, Rabbits, Rats, Recombinant Proteins, Steroid Hydroxylases
Show Abstract · Added February 12, 2015
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.
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17 MeSH Terms
Construction of a human cytochrome P450 1A1: rat NADPH-cytochrome P450 reductase fusion protein cDNA and expression in Escherichia coli, purification, and catalytic properties of the enzyme in bacterial cells and after purification.
Chun YJ, Shimada T, Guengerich FP
(1996) Arch Biochem Biophys 330: 48-58
MeSH Terms: Animals, Base Sequence, Carcinogens, Chromatography, Affinity, Chromatography, DEAE-Cellulose, Cloning, Molecular, Cytochrome P-450 Enzyme System, DNA Primers, Durapatite, Escherichia coli, Humans, Microbial Sensitivity Tests, Molecular Sequence Data, Molecular Weight, Mutagens, NADPH-Ferrihemoprotein Reductase, Plasmids, Polymerase Chain Reaction, Prodrugs, Rats, Recombinant Fusion Proteins, Restriction Mapping, Salmonella typhimurium, Substrate Specificity
Show Abstract · Added March 5, 2014
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.
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24 MeSH Terms
Purification and characterization of recombinant-expressed cytochrome P450 2C3 from Escherichia coli: 2C3 encodes the 6 beta-hydroxylase deficient form of P450 3b.
Richardson TH, Hsu MH, Kronbach T, Barnes HJ, Chan G, Waterman MR, Kemper B, Johnson EF
(1993) Arch Biochem Biophys 300: 510-6
MeSH Terms: Amino Acid Sequence, Animals, Aryl Hydrocarbon Hydroxylases, Base Sequence, Cell Membrane, Chromatography, Chromatography, DEAE-Cellulose, Cloning, Molecular, Cytochrome P-450 Enzyme System, DNA, Durapatite, Escherichia coli, Exons, Hydroxyapatites, Kinetics, Molecular Sequence Data, Oligodeoxyribonucleotides, Plasmids, Polymerase Chain Reaction, Rabbits, Recombinant Fusion Proteins, Recombinant Proteins, Steroid 16-alpha-Hydroxylase, Steroid Hydroxylases
Show Abstract · Added February 12, 2015
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.
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24 MeSH Terms
Purification and partial characterization of rat liver folate binding protein: cytosol I.
Cook RJ, Wagner C
(1982) Biochemistry 21: 4427-34
MeSH Terms: Animals, Carrier Proteins, Chromatography, Affinity, Chromatography, DEAE-Cellulose, Chromatography, Gel, Chromatography, High Pressure Liquid, Cytosol, Folate Receptors, GPI-Anchored, Formyltetrahydrofolates, Glycine N-Methyltransferase, Liver, Molecular Weight, Rats, Receptors, Cell Surface, Tetrahydrofolates, gamma-Glutamyl Hydrolase
Show Abstract · Added January 20, 2015
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.
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16 MeSH Terms
Fractionation of glycoprotein components of the reduced alkylated renal glomerular basement membrane.
Hudson BG, Spiro RG
(1972) J Biol Chem 247: 4239-47
MeSH Terms: Alkylation, Amino Acids, Animals, Basement Membrane, Carbohydrates, Cattle, Chemical Phenomena, Chemistry, Chromatography, DEAE-Cellulose, Chromatography, Gel, Disaccharides, Disulfides, Electrophoresis, Glycoproteins, Hexosamines, Hexoses, Hydroxylysine, Kidney Glomerulus, Molecular Weight, Neuraminic Acids, Oxidation-Reduction, Peptides, Polysaccharides, Sodium Dodecyl Sulfate, Urea
Added December 10, 2013
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25 MeSH Terms
Kynurenine formamidase. Purification and characterization of the adult chicken liver enzyme and immunochemical analyses of the enzyme of developing chicks.
Bailey CG, Wagner C
(1974) J Biol Chem 249: 4439-44
MeSH Terms: Age Factors, Aminohydrolases, Ammonium Sulfate, Animals, Arylformamidase, Centrifugation, Chick Embryo, Chickens, Chromatography, DEAE-Cellulose, Chromatography, Gel, Electrophoresis, Disc, Female, Hot Temperature, Hydrogen-Ion Concentration, Immunodiffusion, Liver, Molecular Weight, Precipitin Tests, Rabbits, Structure-Activity Relationship
Added January 20, 2015
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20 MeSH Terms
Purification and characterization of a periplasmic oligopeptide binding protein from Escherichia coli.
Guyer CA, Morgan DG, Osheroff N, Staros JV
(1985) J Biol Chem 260: 10812-8
MeSH Terms: Bacterial Proteins, Carrier Proteins, Chromatography, DEAE-Cellulose, Chromatography, Gel, Electrophoresis, Polyacrylamide Gel, Escherichia coli, Escherichia coli Proteins, Kinetics, Lipoproteins, Molecular Weight, Oligopeptides, Protein Conformation
Show Abstract · Added March 5, 2014
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.
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12 MeSH Terms