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

被引：1

作者：

Shen Shaochuan ^{[1
,2
]}

Wang Liangyan ^{[1
]}

Sun Zongtao ^{[3
]}

Li Mingfeng ^{[1
]}

Liu Chengzhi ^{[1
]}

Tian Bing ^{[1
]}

Yun Junxian ^{[2
]}

Hua Yuejin ^{[1
]}

机构：

[1] Zhejiang Univ, Inst Nucl Agr Sci, Key Lab Nucl Agr Sci, Chinese Minist Agr & Zhejiang Prov, Hangzhou 310029, Zhejiang, Peoples R China

[2] Zhejiang Univ Technol, Coll Chem Engn & Mat Sci, State Key Lab Breeding Base Green Chem Synth Tech, Hangzhou 310032, Zhejiang, Peoples R China

[3] Zhejiang Acad Agr Sci, Dept Virol & Biotechnol, Hangzhou 310021, Zhejiang, Peoples R China

来源：

CHINESE JOURNAL OF CHEMICAL ENGINEERING | 2013年 / 21卷 / 06期

基金：

中国国家自然科学基金;

关键词：

chromatography; separation; protein; modeling; bacterium; BACTERIUM DEINOCOCCUS; BREAKTHROUGH PERFORMANCE; CAROTENOID BIOSYNTHESIS; FERMENTATION BROTH; POLYMERIC CRYOGELS; OXIDATIVE STRESS; EXCHANGE CRYOGEL; PURIFICATION; BEDS; RADIATION;

D O I：

10.1016/S1004-9541(13)60514-5

中图分类号：

TQ [化学工业];

学科分类号：

0817 ;

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 x 10(-12) m(2). From the capillary-based model, this cryogel presents a slightly wide normal pore (capillary) size distribution with the mean diameter of 55.2 mu m, the standard deviation of 28.0 mu m and the half of skeleton wall thickness of 2.8 mu m. The pore size distribute from about 10 to 141 mu m 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 x 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.

引用

页码：663 / 669

页数：7