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Title
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Chemically and conformationally authentic active domain of human tissue inhibitor of metalloproteinases-2 refolded from bacterial inclusion bodies.
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Authors
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R.A.Williamson,
D.Natalia,
C.K.Gee,
G.Murphy,
M.D.Carr,
R.B.Freedman.
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Ref.
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Eur J Biochem, 1996,
241,
476-483.
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PubMed id
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Abstract
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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 1H-NMR; no significant differences were
found between it and the native protein. Mass analysis of uniformly 13C-labeled
and 15N-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.
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