SCI和EI收录∣中国化工学会会刊

Chinese Journal of Chemical Engineering ›› 2013, Vol. 21 ›› Issue (6): 663-669.DOI: 10.1016/S1004-9541(13)60514-5

• 生物技术与生物工程 • 上一篇    下一篇

Separation of Recombinant Geranylgeranyl Diphosphate Synthase of Deinococcus radiodurans from Expressed Strain Cell Homogenate by Immobilized Metal Affinity Chromatography on a Characterized Monolithic Cryogel Column

沈绍传1,2, 王梁燕1, 孙宗涛3, 李铭峰1, 刘程智1, 田兵1, 贠军贤2, 华跃进1   

  1. 1 Key Laboratory for Nuclear-Agricultural Sciences of Chinese Ministry of Agriculture and Zhejiang Province, Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou 310029, China;
    2 State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology, College of Chemical Engineering and Materials Science, Zhejiang University of Technology, Hangzhou 310032, China;
    3 Department of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
  • 收稿日期:2012-04-05 修回日期:2012-12-30 出版日期:2013-06-28 发布日期:2013-07-03
  • 通讯作者: HUA Yuejin
  • 基金资助:

    Supported by the National Natural Science Foundation of China (30830006, 20876145, 21036005), the International Science & Technology Cooperation Program from the Ministry of Science and Technology of China (1017), the Special Fund for Agroscientific Research in the Public Interest (201103007), the Fundamental Research Funds for the Central Universities and the Natural Science Foundation of Zhejiang Province (Y4080326, Y407366).

Separation of Recombinant Geranylgeranyl Diphosphate Synthase of Deinococcus radiodurans from Expressed Strain Cell Homogenate by Immobilized Metal Affinity Chromatography on a Characterized Monolithic Cryogel Column

SHEN Shaochuan1,2, WANG Liangyan1, SUN Zongtao3, LI Mingfeng1, LIU Chengzhi1, TIAN Bing1, YUN Junxian2, HUA Yuejin1   

  1. 1 Key Laboratory for Nuclear-Agricultural Sciences of Chinese Ministry of Agriculture and Zhejiang Province, Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou 310029, China;
    2 State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology, College of Chemical Engineering and Materials Science, Zhejiang University of Technology, Hangzhou 310032, China;
    3 Department of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
  • Received:2012-04-05 Revised:2012-12-30 Online:2013-06-28 Published:2013-07-03
  • Contact: HUA Yuejin
  • Supported by:

    Supported by the National Natural Science Foundation of China (30830006, 20876145, 21036005), the International Science & Technology Cooperation Program from the Ministry of Science and Technology of China (1017), the Special Fund for Agroscientific Research in the Public Interest (201103007), the Fundamental Research Funds for the Central Universities and the Natural Science Foundation of Zhejiang Province (Y4080326, Y407366).

摘要: Geranylgeranyl diphosphate synthase (GGPPS) plays a key role in the biosynthesis of antioxidative carotenoid from the extremely radioresistant bacterium Deinococcus radiodurans. In this work, the recombinant GGPPS expressed in Escherichia coli by cloning and transforming the gene dr1395 of D. radiodurans was isolated rapidly by an immobilized metal affinity supermacroporous cryogel, i.e., Cu2+-iminodiacetic acid (IDA)-cryogel. The properties of the Cu2+-IDA-cryogel were characterized using capillary-based mathematical model and experimental measurements. The obtained protein samples were analyzed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The porosity of the present Cu2+-IDA-cryogel is 90.4% and the water permeability is 5.04×10-12 m2. From the capillary-based model, this cryogel presents a slightly wide normal pore (capillary) size distribution with the mean diameter of 55.2 μm, the standard deviation of 28.0 μm and the half of skeleton wall thickness of 2.8 μm. The pore size distribute from about 10 to 141 μm and the effective tortuosity of these capillary pores increases from 2.60 to 9.05. The isolation of the GGPPS from cell homogenate can be achieved at the flow velocity of 3.40×10-4 m·s-1 by the Cu2+-IDA-cryogel bed. High-purity GGPPS (about 91.4%) is obtained according to the SDS-PAGE analysis of the elution samples, indicating that the present method is a promising, simple and effective approach to isolate GGPPS from cell homogenate of engineering strains.

关键词: chromatography, separation, protein, modeling, bacterium

Abstract: Geranylgeranyl diphosphate synthase (GGPPS) plays a key role in the biosynthesis of antioxidative carotenoid from the extremely radioresistant bacterium Deinococcus radiodurans. In this work, the recombinant GGPPS expressed in Escherichia coli by cloning and transforming the gene dr1395 of D. radiodurans was isolated rapidly by an immobilized metal affinity supermacroporous cryogel, i.e., Cu2+-iminodiacetic acid (IDA)-cryogel. The properties of the Cu2+-IDA-cryogel were characterized using capillary-based mathematical model and experimental measurements. The obtained protein samples were analyzed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The porosity of the present Cu2+-IDA-cryogel is 90.4% and the water permeability is 5.04×10-12 m2. From the capillary-based model, this cryogel presents a slightly wide normal pore (capillary) size distribution with the mean diameter of 55.2 μm, the standard deviation of 28.0 μm and the half of skeleton wall thickness of 2.8 μm. The pore size distribute from about 10 to 141 μm and the effective tortuosity of these capillary pores increases from 2.60 to 9.05. The isolation of the GGPPS from cell homogenate can be achieved at the flow velocity of 3.40×10-4 m·s-1 by the Cu2+-IDA-cryogel bed. High-purity GGPPS (about 91.4%) is obtained according to the SDS-PAGE analysis of the elution samples, indicating that the present method is a promising, simple and effective approach to isolate GGPPS from cell homogenate of engineering strains.

Key words: chromatography, separation, protein, modeling, bacterium