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Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris
Published on: February 26, 2010
Molecular cloning of monkey P450 1A1 cDNA and expression in yeast
M Komori1, O Kikuchi, M Kitada
1Division of Analytical Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Japan.
Abstract:
Monkey P450 1A1 cDNA (MKah1) was isolated from the lambda gt11 cDNA library of a liver from a 3-methylcholanthrene (3MC)-treated crab-eating monkey using a dog P450 1A1 cDNA fragment as a probe. MKah1 was 2453 bp long and contained an entire coding region for a polypeptide of 512 residues. The nucleotide and deduced amino acid sequences of MKah1 displayed 95% and 94% identity with those of the human P450 1A1 gene, respectively. Even in the 3' noncoding region, MKah1 showed 94% homology with human P450 1A1, whereas it showed less than 69% homology with other mammalian P450 1A1. Monkey P450 1A1 mRNA was not detectable in untreated livers, but was induced by polychlorinated biphenyl and 3MC. The expression plasmid (designated as pMKC-1) was constructed by introduction of the coding region of MKah1 into a yeast expression vector (pAM82) containing the promoter of acid phosphatase (APase). Northern blot analysis revealed that monkey P450 1A1 mRNA was expressed in yeast under the control of the APase promoter. Microsomes from yeast transformed by pMKC-1 catalyzed 7-ethoxycoumarin O-deethylation, benzo(a)pyrene hydroxylation and the mutagenic activation of 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ), 3-amino-1-methyl-5H-pyrido(4,3-b)-indole acetate (Trp-P-2) and 2-amino-6-methyldipyrido(1,2-a:3',2'-d)imidazole acetate (Glu-P-1).
Insights
Researchers isolated monkey P450 1A1 cDNA (MKah1) and found it highly similar to human P450 1A1. Yeast expression of MKah1 demonstrated its enzymatic activity in metabolizing various compounds.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Cytochrome P450 1A1 (CYP1A1) is a crucial enzyme involved in xenobiotic metabolism.
- Understanding species-specific variations in CYP1A1 is important for drug development and toxicology.
- The crab-eating monkey P450 1A1 gene (MKah1) sequence and function have not been extensively characterized.
Purpose of the Study:
- To isolate and characterize the monkey P450 1A1 cDNA (MKah1).
- To investigate the inducibility of monkey P450 1A1 mRNA by environmental chemicals.
- To express functional monkey P450 1A1 in yeast and assess its metabolic capabilities.
Main Methods:
- Isolation of MKah1 cDNA from a 3-methylcholanthrene-treated monkey liver library using a dog P450 1A1 probe.
- Sequence analysis of MKah1 and comparison with human and other mammalian P450 1A1 genes.
- Construction of a yeast expression plasmid (pMKC-1) and transformation of yeast.
- Northern blot analysis to detect MKah1 mRNA expression.
- Enzymatic assays using yeast microsomes to evaluate O-deethylation, hydroxylation, and mutagenic activation activities.
Main Results:
- MKah1 cDNA is 2453 bp, encoding a 512-residue polypeptide with high nucleotide (95%) and amino acid (94%) identity to human P450 1A1.
- MKah1 showed significantly higher homology to human P450 1A1 (94%) than to other mammalian P450 1A1 (<69%) in the 3' noncoding region.
- Monkey P450 1A1 mRNA was undetectable in untreated livers but induced by polychlorinated biphenyl and 3MC.
- Yeast expressing pMKC-1 catalyzed 7-ethoxycoumarin O-deethylation and benzo(a)pyrene hydroxylation.
- Yeast microsomes activated mutagenicity of IQ, Trp-P-2, and Glu-P-1.
Conclusions:
- Monkey P450 1A1 (MKah1) is highly homologous to human P450 1A1, suggesting conserved function.
- MKah1 expression is inducible by specific xenobiotics, similar to other species.
- Yeast expression system successfully produced functional monkey P450 1A1, enabling the study of its catalytic activities.
- The functional characterization of MKah1 in yeast provides a valuable tool for toxicological and pharmacological studies.
