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Title
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The signal transducer gp130--bacterial expression, refolding and properties of the carboxy-terminal domain of the cytokine-binding module.
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Authors
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G.Müller-Newen,
S.Pflanz,
U.Hassiepen,
J.Stahl,
A.Wollmer,
P.C.Heinrich,
J.Grötzinger.
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Ref.
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Eur J Biochem, 1997,
247,
425-431.
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PubMed id
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Abstract
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Gp130 is the signal transducing receptor subunit of the so-called
interleukin-6-type cytokines. This transmembrane protein is a member of the
cytokine-receptor superfamily predicted to consist of six
fibronectin-type-III-like domains in its extracellular part. The second and the
third domain constitute the so-called cytokine-binding module. Domain 2 is
characterized by a set of four conserved Cys residues, domain 3 by a conserved
WSXWS motif. As a first approach to a more detailed characterization of the
cytokine-binding domains of human gp130, we have expressed in Escherichia coli
two forms of domain 3 differing in length. Both proteins were purified and
refolded in a single step applying size-exclusion chromatography. According to
the rotational correlation times deduced from fluorescence anisotropy decay,
they do not form aggregates. CD and fluorescence spectroscopy were used to study
thermal unfolding and denaturation by guanidinium hydrochloride. It was shown
that N- and C-terminal extension by residues of the adjacent hinge regions
substantially increase the thermal stability of the domain, which is conceivable
from a molecular model. These results are the basis for further structural
investigation by NMR spectroscopy.
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