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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2010, Vol. 30 Issue (04): 54-59    DOI: Q786
研究报告     
重组萝卜磷脂氢谷胱甘肽过氧化物酶在毕赤酵母优化表达初步纯化与鉴定
李洋1,李晖2,祝建波1,刘进元2**
1.石河子大学农业生物技术重点实验室 石河子 832000
2.清华大学生命科学学院分子生物学实验室 北京 100084
Optimized Expression and Identification of the Radish Phospholipid Hydroperoxide Glutathione Peroxidase in Pichia pastoris
LI Yang1,LI Hui2,ZHU Jian-bo1,LIU Jin-yuan2
1. Laboratory of Biotechnology, School of Life Sciences, Shihezi University, Shihezi83200, China
2.Laboratory of Molecular Biology, School of Life Sciences，Tsinghua University, Beijing100084, China
 全文: PDF(764 KB)   HTML
摘要：

将萝卜磷脂氢谷胱甘肽过氧化物酶（RsPHGPx）基因插入到分泌表达载体pPIC9K中，转化巴斯德毕赤酵母GS115细胞，筛选具有G418抗性的单拷贝转化子。经过优化表达条件，RsPHGPx在1%甲醇、pH6.0、28℃条件下诱导60h后得到最大表达量，产率约为102 mg/L。通过硫酸铵分级沉淀、脱盐柱脱盐、凝胶过滤等纯化步骤，得到了90%以上纯度的RsPHGPx．活性分析显示纯化获得的RsPHGPx具有依赖于GSH的还原活性, 比活性为4.2μmol/min·mg，为获得大量RsPHGPx而用于应用开发研究奠定了基础。
关键词： 分泌表达;  蛋白纯化;  质谱鉴定;  活性检测    
Abstract:

The expression of RsPHGPx gene in Pichia pastoris was investigated. The RsPHGPx gene inserted into secretory vector pPIC9K was transformed into Pichia pastoris strain GS115.The singlecopy recombinant strains were screened by G418. In addition, the induction conditions were optimized to get the highest expression of the target protein (the optimum:1%methonal,pH6.0,28℃). Finally,it was shown that the recombinant RsPHGPx could be secreted into the culture supernatant to a level of 102mg/L after 60 hours of induction.The fractional ammonium sulfate precipitation, desalination, and gel chromatography were used to purify protein and more than 90% purity of RsPHGPx was obtained. The bioactivity of RsPHGPx was high-dependent redox-active of GSH and reached its max secreted volume at 60h, the specific activity is 4.2μmol/min·mg. The research has laid the foundation for gaining and exploiting a large amount of RsPHGPx.
Key words: Secreted expression    Protein purification    Mass spectrum identification    Analysis of enzymatic activity
收稿日期: 2009-12-30 出版日期: 2010-04-29
基金资助:

国家“863”计划(2007AA100604)、国家自然科学基金(30170080,39770078)、国家重点基础研究发展规划(2006CB101706)资助项目
通讯作者: 刘进元     E-mail: liujy@mail.tsinghua.edu.cn
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李洋
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引用本文:

李洋 李晖 祝建波 刘进元. 重组萝卜磷脂氢谷胱甘肽过氧化物酶在毕赤酵母优化表达初步纯化与鉴定[J]. 中国生物工程杂志, 2010, 30(04): 54-59.

LI Xiang, LI Hui, CHU Jian-Bei, LIU Jin-Yuan. Optimized Expression and Identification of the Radish Phospholipid Hydroperoxide Glutathione Peroxidase in Pichia pastoris. China Biotechnology, 2010, 30(04): 54-59.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/Q786    或    https://manu60.magtech.com.cn/biotech/CN/Y2010/V30/I04/54

［1］ Maiorino M, Maurib P, Roveria A, et al. Primary structure of the nuclear forms of phospholipids hydroperoxide glutathione peroxidase (PHGPx) in rat spermatozoa. FEBS Lett,2005, 579: 667670. 
［2］ Imai H, Nakagawa Y. Biological significance of phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) in mammalian cells. Free Radic Bio Med,2003, 34: 145169. 
［3］ Yang X D, Dong C J, Liu J Y. A plant mitochondrial phospholipid hydroperoxide glutathione peroxidase: its precise localization and higher enzymatic activity. Plant Mol Biol,2006,62:951962. 
［4］ Arai M, Imai H, Koumura T, et al. Mitochondrial phospholipid hydroperoxide glutathione peroxidase plays a major role in preventing oxidative injury to cells. J Biol Chem,1999, 274: 49244933. 
［5］ Kim J G, Parthasarathy S. Oxidation and the spermatozoa. Semin Reprod Endocrinol,1998, 16: 235239. 
［6］ Bourgeron T. Mitochondrial function and infertility. Results Probl Cell Differ,2000, 28: 187210. 
［7］ Imai H, Suzuki K, Ishizaka K, et al. Failure of the expression of phospholipid hydroperoxide glutathione peroxidase in the spermatozoa of human infertile males. Biol Reprod,2001, 64: 674683. 
［8］ Li W J, Gu S, Jin L, et al. A screening of phospholipid hydroperoxide glutathione peroxidase genes in plants. J. Tsinghua Univ (Sci&Tech),2000,40(11): 1214. 
［9］ Yang X D,Li W J,Liu J Y. Isolation and characterization of a novel PHGPx gene in Raphanus sativus. BBAGene Structure and Expression 2005,1728:199205. 
［10］ Yang X D, Liu J Y. Radish phospholipid hydroperoxide glutathione peroxidase gene structure and ppstream regulatory sequence analysis. Prog Biochem Biophysic,2005,32(7):649656. 
［11］ Yang X D, Liu J Y. Micropreparation of a native PHGPx protein from radish seedlings by immunoaffinity chromatography. Prog Biochem Biophysic,2005,32(8):794799. 
［12］ Wan F, Liu J Y. Expression, purification of a recombinant Nterminal 32 amino acids deletion radish PHGPx, Journal of Ji Lin University (Scienc Edition),2008,46: 148153. 
［13］ Li T, Liu G L, Duan M X, et al. Radish phospholipid hydroperoxide glutathione peroxidase provides protection against hydroperoxidemediated injury in mouse 3T3 fibroblasts. Biochemstry and Molecular Biology Reports,2009,46(10):648654. 
［14］ 李甜,段明星,刘进元,等． 萝卜PHGPx对黑色素瘤B16细胞紫外辐射损伤的恢复作用 中国生物工程杂志,2010,30(2):6670. Li T，Duan M X，Liu J Y， et al. China Biotechnology,2010,30(2):6670. 
［15］ Yagi K, Shidoji Y, Komura S, et al. Dissipation of mitochondrialmembrane potential by exogenous phospholipid monohydroperoxide and protection against this effect by transfection of cells with phospholipid hydroperoxide glutathione peroxidase gene. Biochem Biophys Res Commun,1998, 245: 528533. 
［16］ Thiry M, Cingolani D. Optimizing scaleup fermentation processes. Trends Biotechnol, 2002, 20: 103105. 
［17］ Cereghion J L, Cregg J M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiology Reviews,2000, 24(1): 45  66. 
［18］ Barr K A, Hopkins S A, Sreekrishna K. Protocol for efficient secretion of HSA developed from Pichia pastoris. Pharm Eng,1992,12：4851. 
［19］ Gygi S P, Aebersold R. Mass spectrometry and proteomics. Current Opinion in Chemical Biology,2000, 4:489494.
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