Refolding out of guanidine hydrochloride is an effective approach for high-throughput structural studies of small proteins |
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Authors: | Maxwell Karen L Bona Diane Liu Chengsong Arrowsmith Cheryl H Edwards Aled M |
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Institution: | Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada. |
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Abstract: | Low in vivo solubility of recombinant proteins expressed in Escherichia coli can seriously hinder the purification of structural samples for large-scale proteomic NMR and X-ray crystallography studies. Previous results from our laboratory have shown that up to one half of all bacterial and archaeal proteins are insoluble when overexpressed in E. coli. Although a number of strategies may be used to increase in vivo protein solubility, there are no generally applicable methods, and the expression of each insoluble recombinant protein must be individually optimized. For this reason, we have tested a generic denaturation/refolding protein purification procedure to assess the number of structural samples that could be generated by using this methodology. Our results show that a denaturation/refolding protocol is appropriate for many small proteins (
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Keywords: | Protein structure/folding protein solubility protein purification NMR spectroscopy circular dichroism spectroscopy structural proteomics |
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