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.