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Chromatographic methods for the isolation of and refolding of proteins from, Escherichia coli inclusion bodies
Alternative TitleProtein Expr. Purif.
Gu, ZY; Weidenhaupt, M; Ivanova, N; Pavlov, M; Xu, BZ; Su, ZG; Janson, JC
2002-06-01
Source PublicationPROTEIN EXPRESSION AND PURIFICATION
ISSN1046-5928
Volume25Issue:1Pages:174-179
AbstractNew 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).; 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).
KeywordRenaturation Chain
SubtypeArticle
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine
DOI10.1006/prep.2002.1624
URL查看原文
Indexed BySCI
Language英语
WOS KeywordRENATURATION ; CHAIN
WOS Research AreaBiochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
WOS SubjectBiochemical Research Methods ; Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
WOS IDWOS:000176592400022
Citation statistics
Cited Times:54[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Version出版稿
Identifierhttp://ir.ipe.ac.cn/handle/122111/5598
Collection研究所(批量导入)
Affiliation1.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
Recommended Citation
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.
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