Chemically and conformationally authentic active domain of human tissue inhibitor of metalloproteinases-2 refolded from bacterial inclusion bodies

Williamson, Richard A. and Natalia, Dessy and Gee, Christopher K. and Murphy, Gillian and Carr, Mark D. and Freedman, Robert B. (1996) Chemically and conformationally authentic active domain of human tissue inhibitor of metalloproteinases-2 refolded from bacterial inclusion bodies. European Journal of Biochemistry, 241 (2). pp. 476-483. ISSN 0014-2956. (The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided)

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The aggregation of recombinant proteins into inclusion bodies is a major problem for expression in bacterial systems. The inclusion bodies must be solubilized and the denatured protein renatured if an active molecule is to be recovered. We have developed such a procedure for the active N-terminal domain of tissue inhibitor of metalloproteinases-2 [TIMP-2-(1-127)], a small mammalian protein containing three disulfide bonds. Conditions for its renaturation were determined by studying the refolding behaviour of reduced and denatured mammalian-cell-expressed TIMP-(1-127) by intrinsic fluorescence. This strategy allows the development of a refolding protocol before generation of a bacterial expression system, and allows rapid and systematic optimization of each refolding variable by assessing its effect on the rate and extent of the refolding reaction. TIMP-(1-127) was expressed at high levels in Escherichia coli, and refolded from TIMP-2-(1-127) inclusion bodies, by means of the method developed with mammalian-cell-expressed protein, to give a refolding efficiency of 30-40% and a final yield of 11-14 mg purified protein/l culture. The chemical structure and conformation of this material was characterized by electrospray mass spectrometry and two-dimensional H-1-NMR; no significant differences were found between it and the native protein. Mass analysis of uniformly C-13-labeled and N-15-labeled protein was used to help identify a mistranslated TIMP-(1-127) contaminant in the purified refolded sample. This technique provides additional information on the nature of the modification and allows a distinction to be made between those modifications that are cell derived, and those that arise from subsequent handling of the protein.

Item Type: Article
Uncontrolled keywords: tissue inhibitor of metalloproteinase-2; matrix metalloproteinase; protein refolding; mis-translation
Subjects: Q Science > QH Natural history > QH301 Biology
Divisions: Faculties > Science Technology and Medical Studies > School of Biosciences
Depositing User: P. Ogbuji
Date Deposited: 28 Apr 2009 18:26
Last Modified: 16 Jun 2014 14:05
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