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Investigation of protein refolding using a fractional factorial screen: A study of reagent effects and interactions
Journal article   Open access   Peer reviewed

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

Melissa Swope Willis, James K. Hogan, Prakash Prabhakar, Xun Liu, Kuenhi Tsai, Yunyi Wei and Ted Fox
Protein science, Vol.14(7), pp.1818-1826
07/01/2005
DOI: 10.1110/ps.051433205
PMCID: PMC2253366
PMID: 15937284
url
https://europepmc.org/articles/pmc2253366View
Published (Version of record) Open Access

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.
 crystal structure fractional factorial screen high-throughput refolding inclusion bodies protein folding structural genomics

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