Overexpression of a glutamate receptor (GluR2) ligand binding domain in Escherichia coli: application of a novel protein folding screen.
نویسندگان
چکیده
Expression of the S1S2 ligand binding domain [Kuusinen, A., Arvola, M. & Keinänen, K. (1995) EMBO J. 14, 6327-6332] of the rat alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-selective glutamate receptor GluR2 in Escherichia coli under control of a T7 promoter leads to production of >100 mg/liter of histidine-tagged S1S2 protein (HS1S2) in the form of inclusion bodies. Using a novel fractional factorial folding screen and a rational, step-by-step approach, multiple conditions were determined for the folding of the HS1S2 alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid binding domain. Characterization of the HS1S2 ligand binding domain showed that it is water-soluble, monomeric, has significant secondary structure, and is sensitive to trypsinolysis at sites close to the beginning of the putative transmembrane regions. Application of a fractional factorial folding screen to other proteins may provide a useful means to evaluate E. coli as an economical and convenient expression host.
منابع مشابه
A new protein folding screen: application to the ligand binding domains of a glutamate and kainate receptor and to lysozyme and carbonic anhydrase.
Production of folded and biologically active protein from Escherichia coli derived inclusion bodies can only be accomplished if a scheme exists for in vitro naturation. Motivated by the need for a rapid and statistically meaningful method of determining and evaluating protein folding conditions, we have designed a new fractional factorial protein folding screen. The screen includes 12 factors s...
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عنوان ژورنال:
- Proceedings of the National Academy of Sciences of the United States of America
دوره 94 25 شماره
صفحات -
تاریخ انتشار 1997