Refolding of TIMP-2 from Escherichia Coli Inclusion Bodies

Williamson, R.A. (2001) Refolding of TIMP-2 from Escherichia Coli Inclusion Bodies. Methods in Molecular Biology, 151 . pp. 257-265. ISSN 1064-3745. (The full text of this publication is not available from this repository)

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Official URL
http://dx.doi.org/10.1385/1-59259-046-2:257

Abstract

E. coli is a convenient host in which to express recombinant proteins. The technology is available to most laboratories as it is relatively inexpensive and does not require extensive expertise. The major drawback of E. coli as an expression host is the inability of the organism to carry out many posttrans-lational modifications, including glycosylation and disulphide bond formation. High-level intracellular expression of many mammalian proteins in E. coli results in the formation of large insoluble aggregates, known as inclusion bodies (1). These dense bodies consist predominantly of the misfolded recombinant product, together with components of the transcription/translation machinery (i.e., RNA polymerase, ribosomal RNA, and plasmid DNA). The TIMPs are invariably insoluble when expressed in E. coli, their folding requirement for the formation of 6 disulphide bonds being incompatible with the reducing environment of the E. coli cell. Fortunately, active, correctly folded recombinant protein can often be recovered from insoluble inclusion bodies by a process of solubilization and in vitro refolding (2–3). Indeed, inclusion body formation has the advantage that the recombinant product often accumulates to high levels in the cell (up to 30% of total cell protein) and allows easy isolation of that protein, a relatively pure and stable form (inclusion bodies are typically >50% recombinant protein).

Item Type: Article
Additional information: Also published in Methods in Molecular Biology Vol.622, 2010. See KAR ID 31440. 1064-3745 (Print) Journal Article Research Support, Non-U.S. Gov't
Uncontrolled keywords: Animals Escherichia coli/chemistry/genetics Gene Expression Humans Inclusion Bodies/chemistry/genetics Protein Folding Protein Structure, Tertiary Recombinant Proteins/chemistry/genetics/isolation & purification Tissue Inhibitor of Metalloproteinase-2/*chemistry/genetics/isolation & purification
Subjects: Q Science
Divisions: Faculties > Science Technology and Medical Studies > School of Biosciences > Protein Science Group
Depositing User: Richard Williamson
Date Deposited: 30 Sep 2008 18:33
Last Modified: 21 Mar 2014 16:21
Resource URI: http://kar.kent.ac.uk/id/eprint/5317 (The current URI for this page, for reference purposes)
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