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  

作　　者：SHEN Shaochuan WANG Liangyan SUN Zongtao LI Mingfeng LIU Chengzhi TIAN Bing YUN Junxian HUA Yuejin 

机构地区：[1]Key Laboratory for Nuclear-Agricultural Sciences of Chinese Ministry of Agriculture and Zhejiang Province, Instituteof Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou 310029, China [2]State Key Laboratory Breeding Base of Geen 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

出　　处：《Chinese Journal of Chemical Engineering》2013年第6期663-669,共7页中国化学工程学报（英文版）

基　　金：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 recombinantGGPPS 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 experi- mental 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 5.04x10-12 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 3.40x 10-4μm. 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 ef- fective atmroach to isolate GGPPS from cell homoenate of engineering, strains.Geranylgeranyl diphosphate synthase (GGPPS) plays a key role in the biosynthesis of antioxidative ca-rotenoid 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 experi-mental 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 permeabil-ity 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 ef-fective approach to isolate GGPPS from cell homogenate of engineering strains.

关 键 词：chromatography SEPARATION protein modeling bactenum 

分 类 号：Q78[生物学—分子生物学] S124[农业科学—农业基础科学]