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Title
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Overexpression, purification, and refolding of link module from human TSG-6 in Escherichia coli: effect of temperature, media, and mutagenesis on lysine misincorporation at arginine AGA codons.
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Authors
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A.J.Day,
R.T.Aplin,
A.C.Willis.
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Ref.
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Protein Expr Purif, 1996,
8,
1.
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PubMed id
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Abstract
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The Link module, a 98-amino-acid domain found in hyaluronan binding proteins of
human tumor necrosis factor stimulated gene 6 was overexpressed in Escherichia
coli. Electrospray ionization mass spectrometry revealed that only 50% of the
expressed protein had the expected wild-type molecular weight, with the
remaining material having between 1 and 4 arginine to lysine substitutions,
arising due to misincorporation at AGA codons. The level of misincorporation was
almost completely abolished by mutation of the 4 AGA codons to CGT. This
mutation to high-usage arginine codons also increased the level of heterologous
protein expression. Refolding of the Link module, which occurred during the
purification procedure, gave two species with different disulfide bond
organizations that could be separated by high-performance liquid chromatography.
One of these had a disulfide bond arrangement consistent with that found in
other Link modules and, by nuclear magnetic resonance spectroscopy, was shown to
be folded.
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