Regular ArticlePurification and Characterization of Recombinant-Expressed Cytochrome P450 2C3 from Escherichia coli: 2C3 Encodes the 6β-Hydroxylase Deficient Form of P450-3b
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Biophysical characterization of Aptenodytes forsteri cytochrome P450 aromatase
2018, Journal of Inorganic BiochemistryCitation Excerpt :Accordingly, full-length afCYP19 maintains 70%, 72% and 74% identity with the rat, human, and pig orthologs, respectively, although those in the active site are completely conserved (boxed residues in Fig. 1). The afCYP19 gene was modified so that the first 39 amino acids comprising the N-terminal transmembrane domain were replaced with the MAKKTSSKGR sequence (Fig. 1) previously used to facilitate expression of rabbit CYP2C3 and CYP2C11 [29–31]. Four additional histidines were added to the C-terminus for purification by Ni2+-NTA affinity chromatography.
A Workflow for Studying Specialized Metabolism in Nonmodel Eukaryotic Organisms
2016, Methods in EnzymologyCitation Excerpt :For example, P450s, a ubiquitous family of heme-thiolate enzymes widely distributed in eukaryotic natural product biosynthesis, are anchored into the membrane of endoplasmic reticulum (ER) (Werck-Reichhart & Feyereisen, 2000). To enable expression of eukaryotic P450s in E. coli, the native N-terminal ER-membrane anchoring sequence can be replaced with a bacterial lipid bilayer-targeting sequence (Pritchard et al., 1998; Richardson et al., 1993). Alternatively, Saccharomyces cerevisiae has become the preferred system for recombinant eukaryotic P450 expression.
Gene engineering, purification, crystallization and preliminary X-ray diffraction of cytochrome P450 p-coumarate-3-hydroxylase (C3H), the Arabidopsis membrane protein
2011, Protein Expression and PurificationCitation Excerpt :Consequently, C3H is expressed at high levels in E. coli as a modified protein, C3Hmod, where the native N-terminal sequence is replaced with that employed for heterologous expression of mammalian P450s in E. coli, such as P450-2A6, -2B4, -2C5, -2C8 and -2C9 [14–18] (Fig. 2A and B). These changes (deletion, addition, and site-directed mutagenesis) to the N-terminus of C3H retain the hydrophobic character of the putative membrane-spanning segment while incorporating codons that facilitate expression in E. coli [19]. In the present study, a hydrophobic segment in the N-terminus of C3H that is thought to function as a membrane-spanning domain was removed to generate C3Hmod with the aim of preventing incorporation of cytochrome into E. coli membranes following its expression.
Modifications in the N-terminus of an insect cytochrome P450 enhance production of catalytically active protein in baculovirus-Sf9 cell expression systems
2008, Insect Biochemistry and Molecular BiologyCrystal structure of human cytochrome P450 2D6
2006, Journal of Biological ChemistryCitation Excerpt :The heme model was least-squares-fitted to the crystal structure but otherwise was kept rigid during the simulation, thus alleviating the need for special parameters for the octahedral iron complex, which is not readily handled by conventional force fields. Expression of 2D6 Truncates—P450 2D6 expresses at high levels in E. coli if the extreme N terminus containing the putative membranespanning region is replaced with different signal sequences, the OmpA signal sequence (53), or the sequence used to express P450 17α in E. coli (54). However, 2D6 protein generated with these N-terminal sequences associates with E. coli membranes and is not amenable to crystallization.
Engineering microsomal cytochrome P450 2C5 to be a soluble, monomeric enzyme: Mutations that alter aggregation, phospholipid dependence of catalysis, and membrane binding
2000, Journal of Biological ChemistryCitation Excerpt :Reactions were initiated by the addition of 1 mm NADPH. The method used for the separation of [14C]progesterone and its metabolites by thin layer chromatography has been previously reported (16). The amount of product formation was determined using a PhosphorImager SI following exposure of the screens to the thin layer chromatography plates.