Williamson, R.A. and Natalia, C. and Gee, C.K and Murphy, G.H. and Carr, M.D. and Freedman, R.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.
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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.
|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:||20 Apr 2012 14:52|
|Resource URI:||http://kar.kent.ac.uk/id/eprint/18584 (The current URI for this page, for reference purposes)|
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