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J Biomol Tech. 2007 February; 18(1): 76.
PMCID: PMC2291849

P219-T Simultaneous and Systematic Evaluation of 96 Protein Refolding Conditions

Abstract

Proteomics requires large amounts of highly pure and correctly folded protein. This need is often met using heterologous expression systems such as Escherichia coli. Over-expression of foreign proteins in E. coli commonly results in the formation of inclusion bodies—dense, insoluble aggregates of misfolded protein. Though commonly viewed as detrimental, inclusion bodies are easily purified, resistant to proteolysis, and can be solubilized with chaotropic agents. Defining conditions that promote refolding of a chemically solubilized target protein into its native conformation, however, is largely empirical. The chances of identifying an optimal refolding condition can be increased by simultaneously and systematically evaluating a large number of refolding conditions. To meet this need, we have developed the iFOLD Protein Refolding systems, a collection of 96-well plate-based refolding screens. System 1 uses N-lauroylsarcosine, a chaotropic anionic detergent, to denature the inclusion bodies, while System 2 uses either guanidine hydrochloride or urea as the denaturant. With both systems, proteins are refolded by rapid dilution of the denatured inclusion bodies into the 96-well refolding plate. The plate contains 92 (System 1) or 95 (System 2) unique protein refolding solutions, each comprised of buffers, salts, redox couples, and protein refolding additives. Using the systems, we have successfully identified high-yielding refolding conditions for a green fluorescent fusion protein, a mammalian endopeptidase, a matrix metalloprotease, and a viral protease. Refolding was measured using spectrophotometric, fluorescent, and target-specific activity assays. High-yielding refolding conditions identified by the screens have been scaled 10,000-fold from 50 μg to 500 mg. Significantly, all steps of the iFOLD systems are equally compatible with manual use and high-throughput automated liquid-handling systems.


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