A medium or high throughput protein refolding assay

Nathan P. Cowieson, Beth Wensley, Gaufier Robin, Gregor Guncar, Jade Forwood, David Hume, Bostjan Kobe, Jennifer L. Martin

Research output: Book chapter/Published conference paperChapter

4 Citations (Scopus)

Abstract

Expression of insoluble protein in E. coli is a major bottleneck of high throughput structural biology projects. Refolding proteins into native conformations from inclusion bodies could significantly increase the number of protein targets that can be taken on to structural studies. This chapter presents a simple assay for screening insoluble protein targets and identifying those that are most amenable to refolding. The assay is based on the observation that when proteins are refolded while bound to metal affinity resin, misfolded proteins are generally not eluted by imidazole. This difference is exploited here to distinguish between folded and misfolded proteins. Two implementations of the assay are described. The assay fits well into a standard high throughput structural biology pipeline, because it begins with the inclusion body preparations that are a byproduct of small-scale, automated expression and purification trials and does not require additional facilities. Two formats of the assay are described, a manual assay that is useful for screening small numbers of targets, and an automated implementation that is useful for large numbers of targets.
Original languageEnglish
Title of host publicationMethods in Molecular Biology Structural Genomics (series)
Place of PublicationTotowa
PublisherHumana Press
Pages269-275
Number of pages7
Volume426
Edition17
ISBN (Print)9781588298096
DOIs
Publication statusPublished - 2008

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    Cowieson, N. P., Wensley, B., Robin, G., Guncar, G., Forwood, J., Hume, D., Kobe, B., & Martin, J. L. (2008). A medium or high throughput protein refolding assay. In Methods in Molecular Biology Structural Genomics (series) (17 ed., Vol. 426, pp. 269-275). Humana Press. https://doi.org/10.1007/978-1-60327-058-8_17