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

Vertex Pharmaceuticals, Cambridge, Massachusetts 02139, USA

(RECEIVED February 25, 2005; FINAL REVISION February 25, 2005; ACCEPTED March 28, 2005)

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; high-throughput refolding; structural genomics

Abbreviations: AMP-PNP, 5'-adenylylimidodiphosphate • ME, -mercaptoethanol • BMC, bis-mercaptoacetamide cyclohexane • Cdc25A, cell division cycle 25A phosphatase • DDM, n-dodecyl--D-maltopyranoside • DTT, dithiothreitol • DYRK3, dual specificity Yak1-related kinase 3 • GdnHCl, guanidine hydrochloride • GSH, reduced glutathione • GSSG, oxidized glutathione • ICE, interleukin-1 converting enzyme • IMPDH, inosine 5'-monophosphate dehydrogenase • LDH, lactate dehydrogenase • MAPKAP-K5, mitogen-activated protein kinase-activated protein kinase 5 • MES, 2-[N-morpholino] ethanesulfonic acid • NADH, -nicotinamide-adenine-dinucleotide, reduced • NADP⁺, -nicotinamide-adenine-dinucleotide phosphate • NDSB, nondetergent sulfobetaine • PEG 3350, polyethylene glycol 3350 Da • pNPP, p-nitrophenylphosphate • RNase A, ribonuclease A • TCEP, tris(2-carboxyethylphosphine) • Tris-HCl, tris (hydroxymethyl)aminomethane hydrochloride • Tween 80, polyoxyethylene (80) sorbitan monolaurate

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051433205.

Reprint requests to: Ted Fox, Vertex Pharmaceuticals, 130 Waverly Street, Cambridge, MA 02139, USA; e-mail: ted_fox{at}vrtx.com; fax: (617) 444-6820.

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