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题名: Establishment of a yeast system that stably expresses human cytochrome P450 reductase: Application for the study of drug metabolism of cytochrome P450s in vitro
作者: Cheng, J;  Wan, DF;  Gu, JR;  Gong, Y;  Yang, SL;  Hao, DC;  Yang, L
关键词: cytochrome P450 ;  human cytochrome P450 reductase ;  human cytochrome P450-3A4 ;  PGK promoter ;  stable expression
刊名: PROTEIN EXPRESSION AND PURIFICATION
发表日期: 2006-06-01
DOI: 10.1016/j.pep.2005.11.022
卷: 47, 期:2, 页:467-476
收录类别: SCI
文章类型: Article
WOS标题词: Science & Technology ;  Life Sciences & Biomedicine
类目[WOS]: Biochemical Research Methods ;  Biochemistry & Molecular Biology ;  Biotechnology & Applied Microbiology
研究领域[WOS]: Biochemistry & Molecular Biology ;  Biotechnology & Applied Microbiology
英文摘要: 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 BWG1-7 alpha 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 443.2 nmol 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 35.53 pmol/mg, and the 6 beta-hydroxylation testosterone formation activity of hCYP3A4 expressed was 7.5 nmol/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. (c) 2005 Elsevier Inc. All rights reserved.
关键词[WOS]: SACCHAROMYCES-CEREVISIAE ;  NADPH-CYTOCHROME-P-450 REDUCTASE ;  MAMMALIAN CYTOCHROME-P450 ;  HETEROLOGOUS EXPRESSION ;  LIVER-MICROSOMES ;  P-450 REDUCTASE ;  KINASE GENE ;  NADPH ;  ENZYME ;  B(5)
语种: 英语
WOS记录号: WOS:000238277000018
Citation statistics: 
内容类型: 期刊论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/140189
Appears in Collections:中国科学院大连化学物理研究所_期刊论文

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作者单位: 1.Chinese Acad Sci, Dalian Inst Chem Phys, Lab Pharmaceut Resource Discovery, Dalian 116023, Peoples R China
2.Chinese Acad Sci, Grad Sch, Beijing 100864, Peoples R China
3.Shanghai Canc Inst, Shanghai 200000, Peoples R China
4.Chinese Acad Sci, Shanghai Res Ctr Bioengn, Shanghai 200233, Peoples R China

Recommended Citation:
Cheng, J,Wan, DF,Gu, JR,et al. Establishment of a yeast system that stably expresses human cytochrome P450 reductase: Application for the study of drug metabolism of cytochrome P450s in vitro[J]. PROTEIN EXPRESSION AND PURIFICATION,2006,47(2):467-476.
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