Investigation of protein refolding using a fractional factorial screen: A study of reagent effects and interactions

Abstract

A recurring obstacle for structural genomics is the expression of insoluble, aggregated proteins. In these cases, the use of alternative salvage strategies, like in vitro refolding, is hindered by the lack of a universal refolding method. To overcome this obstacle, fractional factorial screens have been introduced as a systematic and rapid method to identify refolding conditions. However, methodical analyses of the effectiveness of refolding reagents on large sets of proteins remain limited. In this study, we address this void by designing a fractional factorial screen to rapidly explore the effect of 14 different reagents on the refolding of 33 structurally and functionally diverse proteins. The refolding data was analyzed using statistical methods to determine the effect of each refolding additive. The screen has been miniaturized for automation resulting in reduced protein requirements and increased throughput. Our results show that the choice of pH and reducing agent had the largest impact on protein refolding. Bis-mercaptoacetamide cyclohexane (BMC) and tris(2-carboxyethylphosphine) (TCEP) were superior reductants when compared to others in the screen. BMC was particularly effective in refolding disulfide-containing proteins, while TCEP was better for nondisulfide-containing proteins. From the screen, we successfully identified a positive synergistic interaction between nondetergent sulfobetaine 201 (NDSB 201) and BMC on Cdc25A refolding. The soluble protein resulting from this interaction crystallized and yielded a 2.2 Å structure. Our method, which combines a fractional factorial screen with statistical analysis of the data, provides a powerful approach for the identification of optimal refolding reagents in a general refolding screen. Keywords: protein folding; fractional factorial screen; crystal structure; inclusion bodies; highthroughput refolding; structural genomicsThe identification of 20,000-25,000 genes from the human genome project has resulted in a wealth of potential targets for structural biology investigation and pharmaceutical design (International Human Genome Sequencing Consortium 2004). Since the completion of the project, expectations have been high that the number of protein crystal structures would dramatically increase but, in reality, there has only been a moderate rise in the number of crystal structures, due largely to a lack of sufficient quantities of protein suitable for structural studies (Service 2002). Although the technology responsible for expressing recombinant proteins is highly developed , it is still difficult to produce enough soluble protein for these structural studies. The ultimate goal of determining crystal structures on a genome-wide scale requires methods designed to improve the yield of functional protein.Reprint requests to: Ted Fox, Vertex Pharmaceuticals, 130 Waverly Street, Cambridge, MA 02139, USA; e-mail: ted_fox@vrtx.com; fax: (617) 444-6820.Abbreviations: AMP-PNP, 5 0 -adenylylimidodiphosphate; bME, b-mercaptoethanol; BMC, bis-mercaptoacetamide...

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