Optimized Expression and Identification of the Radish Phospholipid Hydroperoxide Glutathione Peroxidase in Pichia pastoris

doi:Q786

China Biotechnology

2010, Vol. 30

Issue (04): 54-59 DOI: Q786

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

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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

Received: 30 December 2009 Published: 29 April 2010

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Cite this article:

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.

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

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