Abstract
Here, we describe a simple, rapid, cost-effective, and efficient novel one-step purification method for GST-tagged peptides and small proteins. This novel technique applies to proteins and peptides that are known to be thermally stable at 60 °C and do not have elaborate structure(s) and whose heat-induced unfolding is reversible. This method takes advantage of glutathione S-transferase from Schistosoma japonicum (sj26GST) precipitating when heated at 60 °C. Purified GST-fusion products are subjected to enzymatic cleavage to separate the GST tag from the target peptide or small proteins. In our proposed method, the cleavage products are heated at 60 °C for 20 min which results in the precipitation of the GST tag. Subsequently, the GST tag is separated from the target peptide or small protein by high-speed centrifugation. Biophysical experiments such as SDS-PAGE, circular dichroism, isothermal titration calorimetry, mass spectroscopy, and multidimensional NMR spectroscopy confirm that the target peptides and small proteins are purified to more than 95% homogeneity, intact native conformation, and no significant change in the binding affinity of heat-treated purified product to the interacting partners.
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Acknowledgments
This work was supported by the Department of Energy (DE-FG02- 01ER15161), the National Institutes of Health/National Cancer Institute NIH/NCI (1 RO1 CA 172631) and the NIH through the COBRE program (P30 GM103450), and the Arkansas Biosciences Institute (ABI-TKSK- 2016-17). This work was also supported by the National Institute of General Medical Sciences of the National Institutes of Health under award P20GM139768 and the Arkansas Integrative Metabolic Research Center at the University of Arkansas.
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Okoto, P.S., Sonniala, S., Sakhel, B., Muhoza, D., Adams, P., Kumar, T.K.S. (2023). A Simple Purification Method for Heat-Stable Recombinant Low Molecular Weight Proteins and Peptides Via GST-Fusion Products. In: Sousa, Â., Passarinha, L. (eds) Advanced Methods in Structural Biology. Methods in Molecular Biology, vol 2652. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3147-8_8
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DOI: https://doi.org/10.1007/978-1-0716-3147-8_8
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