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

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    Authors
    Gu, Zhenyu
    Weidenhaupt, Marianne
    Ivanova, Natalia
    Pavlov, Michail
    Xu, Bingze
    Su, Zhi-Guo
    Janson, Jan-Christer
    UMass Chan Affiliations
    Department of Biochemistry and Molecular Pharmacology
    Document Type
    Journal Article
    Publication Date
    2002-06-20
    Keywords
    Centrifugation
    Chemistry Techniques, Analytical
    Chromatography
    Chromatography, Gel
    Chromatography, Ion Exchange
    Cloning, Molecular
    Escherichia coli
    Hydrogen-Ion Concentration
    Inclusion Bodies
    Models, Genetic
    Protein Folding
    Urea
    Biochemistry, Biophysics, and Structural Biology
    Pharmacology, Toxicology and Environmental Health
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    Link to Full Text
    http://dx.doi.org/10.1006/prep.2002.1624
    Abstract
    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.
    Source
    Protein Expr Purif. 2002 Jun;25(1):174-9. Link to article on publisher's site
    DOI
    10.1006/prep.2002.1624
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/25988
    PubMed ID
    12071713
    Related Resources
    Link to Article in PubMed
    ae974a485f413a2113503eed53cd6c53
    10.1006/prep.2002.1624
    Scopus Count
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