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.
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Acknowledgments
The authors thank Mareike Kurz, Christine Gee, Thomas Huber, Timothy Ravasi, Munish Puri, and Lynn Pauron for research support. This work was supported by a University of Queensland Postdoctoral Fellowship and an Australian Synchrotron Research Program Fellowship (to N. C. P.) and by an Australian Research Council (ARC) grant to J. L. M. and B. K. B. K. is an ARC Federation Fellow and a National Health and Medical Research Council (NHMRC) Honorary Research Fellow.
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Cowieson, N.P. et al. (2008). A Medium or High Throughput Protein Refolding Assay. In: Kobe, B., Guss, M., Huber, T. (eds) Structural Proteomics. Methods in Molecular Biology™, vol 426. Humana Press. https://doi.org/10.1007/978-1-60327-058-8_17
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