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
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- 作者：Shaochuan SHEN ; Liangyan WANG ; Zongtao SUN ; Mingfeng LI ; Chengzhi LIU ; Bing TIAN ; Junxian YUN ; Yuejin HUA
- 关键词：chromatography ; separation ; protein ; modeling ; bacterium
- 刊名：Chinese Journal of Chemical Engineering
- 出版年：2013
- 出版时间：June, 2013
- 年：2013
- 卷：21
- 期：6
- 页码：663-669
- 全文大小：695 K
文摘
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¡Á10?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 ¦Ìm, the standard deviation of 28.0 ¦Ìm and the half of skeleton wall thickness of 2.8 ¦Ìm. The pore size distribute from about 10 to 141 ¦Ìm 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¡Á10?4 m¡¤?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.