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Establishment of a yeast system that stably expresses human cytochrome P450 reductase: Application for the study of drug metabolism of cytochrome P450s in vitro
- Source :
-
Protein Expression & Purification . Jun2006, Vol. 47 Issue 2, p467-476. 10p. - Publication Year :
- 2006
-
Abstract
- Abstract: Cytochrome P450s (CYPs) hold a balance in studying pharmacokinetics, toxico-kinetics, drug metabolism, and drug–drug interactions, which require association with cytochrome P450 reductase (CPR) to achieve optimal activity. A novel system of Saccharomyces cerevisiae useful for expression studies of mammalian microsomal CYPs was established. Human CPR (hCPR) was co-expressed with human CYP3A4 (hCYP3A4) in this system, and two expression plasmids pTpLC and pYeplac195-3A4 containing the cDNA of hCPR and hCYP3A4 were constructed, respectively. The two plasmids were applied first and controlled by phosphoglycerate kinase (PGK) promoter. S. cerevisiae BWG1-7α transformed with the expression plasmids produced the respective proteins in the expected molecular sizes reactive with both anti-hCYP3A4 immunoglobulin (Ig) and anti-hCPR Ig. The activity of hCPR in yeast BWG-CPR was 443.2nmol reduced cytochrome c/min/mg, which was about three times the CPR activity of the microsome prepared from the parental yeast. The protein amount of hCYP3A4 in BWG-CPR/3A4 was 35.53pmol/mg, and the 6β-hydroxylation testosterone formation activity of hCYP3A4 expressed was 7.5nmol/min/nmol CYP, 30 times higher than the activity of hCYP3A4 expressed in the parental yeast, and almost two times the activity of hCYP3A4 from homologous human liver microsome. Meanwhile, BWG-CPR/3A4 retained 100 generations under nonselective culture conditions, indicating this yeast was a mitotically stable transformant. BWG-CPR was further tested daily by the PCR amplification of hCPR of yeast genome, Western blot analysis, and the activity assay of hCPR of yeast microsome. This special expression host for CYPs was validated to be stable and efficient for the expression of CYPs, applying as an effective selection model for the drug metabolism in vitro. [Copyright &y& Elsevier]
- Subjects :
- *YEAST
*LEAVENING agents
*DRUG metabolism
*BIOCHEMISTRY
Subjects
Details
- Language :
- English
- ISSN :
- 10465928
- Volume :
- 47
- Issue :
- 2
- Database :
- Academic Search Index
- Journal :
- Protein Expression & Purification
- Publication Type :
- Academic Journal
- Accession number :
- 20901088
- Full Text :
- https://doi.org/10.1016/j.pep.2005.11.022