ReBaTSA: A simplified CeTSA protocol for studying recombinant mutant proteins in bacterial extracts

Introduction: The study of protein stability is crucial to biochemistry and relies on different methodologies. Recently, the Cellular Thermal Shift Assay has been introduced to study protein stability in whole cells. Methods: We report a novel application of CeTSA named ReBaTSA. This Recombinant Bacterial TSA was performed using clear extracts from bacteria expressing a recombinant protein, incubated at different temperatures, centrifuged and analyzed via SDS-PAGE. Results and conclusions: We demonstrated the feasibility and reliability of this simplified approach. We validated the method using the protein phosphomannomutase-2 and its common mutants, which were compared in the presence or the absence of a known ligand.