Regular ArticleEscherichia coli Expression and Characterization of Cytochromes P450 2B11, 2B1, and 2B5
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Optimizations to achieve high-level expression of cytochrome P450 proteins using Escherichia coli expression systems
2013, Protein Expression and PurificationCitation Excerpt :It is important to note the host strain selection also influences expression. In optimizing the expression conditions of CYP2B11, the use of XL-1 cells yielded the highest protein level (200 nmol/L) while JM105 the lowest (128 nmol/L) [44]. Similarly, in expressing CYP2C10 it was shown that no expression occurred in JM105 cells, but transformation in DH5-alpha cells allowed protein expression [31].
Bioengineering of isoquinoline alkaloid production in microbial systems
2013, Advances in Botanical ResearchCitation Excerpt :Since then, there have been various attempts to functionally express heterologous P450 genes after altering their N-terminal region. The most common strategies have been to exchange the N-terminal sequence with the sequence ‘MALLAVF’, which was used in CYP17A expression (John, Hasler, He, & Halpert, 1994) and to eliminate the hydrophobic region from the N-terminal region (Iwata et al., 1998; von Wachenfeldt, Richardson, Cosme, & Johnson, 1997). In addition, processing can be optimised by fusing P450 genes with signal sequences from E. coli, such as the leader sequences pelB (pectate lyase B) and ompA (outer membrane protein A) because most eukaryotic P450 oxidoreductases exist as membrane-binding proteins in plant cells (Pritchard et al., 1997).
Role of subunit interactions in P450 oligomers in the loss of homotropic cooperativity in the cytochrome P450 3A4 mutant L211F/D214E/F304W
2007, Archives of Biochemistry and BiophysicsCitation Excerpt :Reactions were initiated by the addition of 1 mM NADPH (Sigma Chemical CO) and stopped after 8 min by the addition of 50 μl of tetrahydrofuran. Quantification of the metabolites by thin-layer chromatography, autoradiography, and liquid scintillation counting was performed as described previously [62]. SPECTRALAB software package [58] was used to fit the data sets to the Hill or Michaelis–Menten equation.
Determination of 3-keto-4-ene steroids and their hydroxylated metabolites catalyzed by recombinant human cytochrome P450 1B1 enzyme using gas chromatography-mass spectrometry with trimethylsilyl derivatization
2003, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life SciencesA truncation of 2B subfamily cytochromes P450 yields increased expression levels, increased solubility, and decreased aggregation while retaining function
2001, Archives of Biochemistry and Biophysics