Chromatographic methods for the isolation of and refolding of proteins from, Escherichia coli inclusion bodies

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	Chromatographic methods for the isolation of and refolding of proteins from, Escherichia coli inclusion bodies
	Gu, ZY ; Weidenhaupt, M ; Ivanova, N ; Pavlov, M ; Xu, BZ ; Su, ZG ; Janson, JC
刊名	PROTEIN EXPRESSION AND PURIFICATION
	2002-06-01
卷号	25 期号:1 页码:174-179
关键词	renaturation chain
ISSN号	1046-5928
其他题名	Protein Expr. Purif.
中文摘要	New methods for the chromatographic isolation of inclusion bodies directly from crude Escherichia coli homogenates and for the refolding of denatured protein are presented. The traditional method of differential centrifugation for the isolation of purified inclusion bodies is replaced by a single gel-filtration step. The principle is that the exclusion limit of the gel particles is chosen such that only the inclusion bodies are excluded, i.e., all other components of the crude homogenate penetrate the gel under the conditions selected. In the novel column refolding process, a decreasing gradient of denaturant (urea or Gu-HCl), combined with an increasing pH gradient, is introduced into a gel-filtration column packed with a gel medium that has an exclusion limit lower than the molecular mass of the protein to be refolded. A limited sample volume of the protein, dissolved in the highest denaturant concentration at the lowest pH of the selected gradient combination, is applied to the column. During the course of elution, the zone of denatured protein moves down the column at a speed approximately threefold higher than that of the denaturant. This means that the protein sample will gradually pass through areas of increasingly lower denaturant concentrations and higher pH, which promotes refolding into the native conformation. The shape and slope of the gradients, as well as the flow rate, will influence the refolding rate and can be adjusted for different protein samples. The principle is illustrated using a denatured recombinant scFv fusion protein obtained from E. coli inclusion bodies. (C) 2002 Elsevier Science (USA).
英文摘要	New methods for the chromatographic isolation of inclusion bodies directly from crude Escherichia coli homogenates and for the refolding of denatured protein are presented. The traditional method of differential centrifugation for the isolation of purified inclusion bodies is replaced by a single gel-filtration step. The principle is that the exclusion limit of the gel particles is chosen such that only the inclusion bodies are excluded, i.e., all other components of the crude homogenate penetrate the gel under the conditions selected. In the novel column refolding process, a decreasing gradient of denaturant (urea or Gu-HCl), combined with an increasing pH gradient, is introduced into a gel-filtration column packed with a gel medium that has an exclusion limit lower than the molecular mass of the protein to be refolded. A limited sample volume of the protein, dissolved in the highest denaturant concentration at the lowest pH of the selected gradient combination, is applied to the column. During the course of elution, the zone of denatured protein moves down the column at a speed approximately threefold higher than that of the denaturant. This means that the protein sample will gradually pass through areas of increasingly lower denaturant concentrations and higher pH, which promotes refolding into the native conformation. The shape and slope of the gradients, as well as the flow rate, will influence the refolding rate and can be adjusted for different protein samples. The principle is illustrated using a denatured recombinant scFv fusion protein obtained from E. coli inclusion bodies. (C) 2002 Elsevier Science (USA).
WOS标题词	Science & Technology ; Life Sciences & Biomedicine
类目[WOS]	Biochemical Research Methods ; Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
研究领域[WOS]	Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
关键词[WOS]	RENATURATION ; CHAIN
收录类别	SCI
原文出处	://WOS:000176592400022
语种	英语
WOS记录号	WOS:000176592400022
公开日期	2013-11-12
内容类型	期刊论文
版本	出版稿
源URL	[http://ir.ipe.ac.cn/handle/122111/5598]
专题	过程工程研究所_研究所（批量导入）
作者单位	1.Chinese Acad Sci, Inst Proc Engn, Beijing 100080, Peoples R China 2.CEA, CNRS, UJF, Lab Ingn Macromol,Inst Biol Struct Jean Pierre Eb, F-38027 Grenoble 1, France 3.Univ Uppsala, Dept Cell & Mol Biol, S-75123 Uppsala, Sweden 4.Ctr Surface Biotechnol, S-75123 Uppsala, Sweden
推荐引用方式 GB/T 7714	Gu, ZY,Weidenhaupt, M,Ivanova, N,et al. Chromatographic methods for the isolation of and refolding of proteins from, Escherichia coli inclusion bodies[J]. PROTEIN EXPRESSION AND PURIFICATION,2002,25(1):174-179.
APA	Gu, ZY.,Weidenhaupt, M.,Ivanova, N.,Pavlov, M.,Xu, BZ.,...&Janson, JC.(2002).Chromatographic methods for the isolation of and refolding of proteins from, Escherichia coli inclusion bodies.PROTEIN EXPRESSION AND PURIFICATION,25(1),174-179.
MLA	Gu, ZY,et al."Chromatographic methods for the isolation of and refolding of proteins from, Escherichia coli inclusion bodies".PROTEIN EXPRESSION AND PURIFICATION 25.1(2002):174-179.