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Refolding out of guanidine hydrochloride is an effective approach for high‐throughput structural studies of small proteins
Author(s) -
Maxwell Karen L.,
Bona Diane,
Liu Chengsong,
Arrowsmith Cheryl H.,
Edwards Aled M.
Publication year - 2003
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.0393503
Subject(s) - denaturation (fissile materials) , escherichia coli , heteronuclear single quantum coherence spectroscopy , chemistry , solubility , in vivo , guanidine , recombinant dna , protein folding , biochemistry , structural genomics , protein tag , chromatography , protein structure , biophysics , nuclear magnetic resonance spectroscopy , biology , fusion protein , stereochemistry , organic chemistry , microbiology and biotechnology , nuclear chemistry , gene
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 (≤18 kD) that are normally soluble in vivo. In addition, refolding the purified proteins by using dialysis against a single buffer allowed us to obtain soluble protein samples of 58% of small proteins that were found in the insoluble fraction in vivo, and 10% of the initial number of proteins provided good heteronuclear single quantum coherence (HSQC) NMR spectra. We conclude that a denaturation/refolding protocol is an efficient way to generate structural samples for high‐throughput studies of small proteins.