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Construction of Salmonella typhimurium YG7108 strains, each coexpressing a form of human cytochrome P450 with NADPH–cytochrome P450 reductase
Author(s) -
Fujita Kenichi,
Nakayama Kazuo,
Yamazaki Yoshiyuki,
Tsuruma Kazuhiro,
Yamada Masami,
Nohmi Takehiko,
Kamataki Tetsuya
Publication year - 2001
Publication title -
environmental and molecular mutagenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1
H-Index - 87
eISSN - 1098-2280
pISSN - 0893-6692
DOI - 10.1002/em.10034
Subject(s) - cytochrome p450 , cyp1a2 , heterologous expression , microsome , biology , cyp3a4 , reductase , biochemistry , enzyme , cytochrome , cyp1b1 , salmonella , cytochrome p450 reductase , isozyme , microbiology and biotechnology , gene , recombinant dna , genetics , cytochrome b , bacteria , mitochondrial dna
A series of Salmonella typhimurium ( S. typhimurium ) YG7108 strains, each coexpressing a form of human cytochrome P450 (CYP) (CYP1A1, CYP1A2, CYP1B1, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, or CYP3A5) together with human NADPH–cytochrome P450 reductase (OR), was established. The parental S. typhimurium YG7108, derived from TA1535, lacks two O 6 ‐methylguanine‐DNA methyltransferase genes, ada and ogt, and is highly sensitive to the mutagenicity of alkylating agents. The expression levels of CYP holo‐protein in the genetically engineered S. typhimurium YG7108 cells, determined by carbon monoxide (CO) difference spectra, ranged from 62 nmol/L culture for CYP2C19 to 169 nmol/L culture for CYP3A4. The expression level of the OR varied, depending on the form of CYP coexpressed, and ranged from 214 to 1029 units/L culture. Each form of CYP expressed in the S. typhimurium YG7108 cells catalyzed the oxidation of a representative substrate at an efficient rate. The rates appeared comparable to the reported activities of CYP expressed in human liver microsomes or CYP in other heterologous systems, indicating that the OR was sufficiently expressed to support the catalytic activity of CYP. These S. typhimurium strains may be useful not only for predicting the metabolic activation of promutagens catalyzed by human CYP but also for identifying the CYP form involved. Environ. Mol. Mutagen. 38:329–338, 2001. © 2001 Wiley‐Liss, Inc.