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高等学校化学研究 ›› 2011, Vol. 27 ›› Issue (5): 813-819.

• Articles • 上一篇    下一篇

Novel Selenium-containing Human Single-chain Variable Fragment with Glutathione Peroxidase Activity from Computer-aided Molecular Design

WANG Cheng1,3, WAN Pei1, GONG Ping-sheng1, LÜ Li-min1, XU Ya-wei1, ZHAO Yang1, HE Bo1, ZHAO Gang1, YAN Gang-lin1*, MU Ying1,2*, LÜ Shao-wu1 and LUO Gui-min1   

  1. 1. Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education, College of Life Science, Jilin University, Changchun 130012, P. R. China;
    2. State Key Laboratory of Industrial Control Technology, Research Center for Analytical Instrumentation, Institute of Cyber-systems and Control, Zhejiang University, Hangzhou 310058, P. R. China;
    3. Department of Biochemistry, School of Basic Medical Sciences, Jilin Medical College, Jilin 132013, P. R. China
  • 收稿日期:2011-05-27 修回日期:2011-06-24 出版日期:2011-09-25 发布日期:2011-09-06
  • 通讯作者: YAN Gang-lin;MU Ying E-mail:ygl@jlu.edu.cn; ymu100@yahoo.com.cn
  • 基金资助:

    Supported by the National Natural Science Foundation of China(No.30970608), the Applicative Technological Project of Bureau of Science and Technology of Changchun City, China(No.2009045), the Development and Planning Major Program of Jilin Provincial Science and Technology Department, China(No.20100948) and the Innovation Method Fund of China (No.2008IM040800).

Novel Selenium-containing Human Single-chain Variable Fragment with Glutathione Peroxidase Activity from Computer-aided Molecular Design

WANG Cheng1,3, WAN Pei1, GONG Ping-sheng1, LÜ Li-min1, XU Ya-wei1, ZHAO Yang1, HE Bo1, ZHAO Gang1, YAN Gang-lin1*, MU Ying1,2*, LÜ Shao-wu1 and LUO Gui-min1   

  1. 1. Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education, College of Life Science, Jilin University, Changchun 130012, P. R. China;
    2. State Key Laboratory of Industrial Control Technology, Research Center for Analytical Instrumentation, Institute of Cyber-systems and Control, Zhejiang University, Hangzhou 310058, P. R. China;
    3. Department of Biochemistry, School of Basic Medical Sciences, Jilin Medical College, Jilin 132013, P. R. China
  • Received:2011-05-27 Revised:2011-06-24 Online:2011-09-25 Published:2011-09-06
  • Contact: YAN Gang-lin;MU Ying E-mail:ygl@jlu.edu.cn; ymu100@yahoo.com.cn
  • Supported by:

    Supported by the National Natural Science Foundation of China(No.30970608), the Applicative Technological Project of Bureau of Science and Technology of Changchun City, China(No.2009045), the Development and Planning Major Program of Jilin Provincial Science and Technology Department, China(No.20100948) and the Innovation Method Fund of China (No.2008IM040800).

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摘要： In order to enhance the glutathione peroxidase(GPX) catalytic activity of the selenium-containing single-chain variable fragments(Se-scFv), a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis, three-dimensional(3D) model building, binding-site analysis and docking. The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b(+), then the scFv protein was expressed in soluble form in Escherichia coli BL21(DE3) and purified by Ni2+-immobilized metal affinity chromatography(IMAC). The serine residue of scFv in the active site was converted into selenocysteine(Sec) with the chemical modification method, thus, the human Se-scFv with GPX activity was obtained. The GPX activity of the Se-scFv protein was characterized. Compared with other Se-scFv, the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione. It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mecha- nism.

关键词: Glutathione peroxidase(GPX), Single-chain variable fragment(scFV), Three-dimensional model, Selenium

Abstract: In order to enhance the glutathione peroxidase(GPX) catalytic activity of the selenium-containing single-chain variable fragments(Se-scFv), a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis, three-dimensional(3D) model building, binding-site analysis and docking. The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b(+), then the scFv protein was expressed in soluble form in Escherichia coli BL21(DE3) and purified by Ni2+-immobilized metal affinity chromatography(IMAC). The serine residue of scFv in the active site was converted into selenocysteine(Sec) with the chemical modification method, thus, the human Se-scFv with GPX activity was obtained. The GPX activity of the Se-scFv protein was characterized. Compared with other Se-scFv, the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione. It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mecha- nism.

Key words: Glutathione peroxidase(GPX), Single-chain variable fragment(scFV), Three-dimensional model, Selenium