A Simple Purification Method for Heat-Stable Recombinant Low Molecular Weight Proteins and Peptides Via GST-Fusion Products.
by Okoto, Patience Salvalina; Sonniala, Shivakumar; Sakhel, Beatrice; Muhoza, Djamali; Adams, Paul; Kumar, Thallapuranam Krishnaswamy Suresh
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.