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.

Exploring ligand interactions with human phosphomannomutases using recombinant bacterial thermal shift assay and biochemical validation.

PMM2-CDG, a disease caused by mutations in phosphomannomutase-2, is the most common congenital disorder of glycosylation. Yet, it still lacks a cure. Targeting phosphomannomutase-2 with pharmacological chaperones or inhibiting the phosphatase activity of phosphomannomutase-1 to enhance intracellular glucose-1,6-bisphosphate have been proposed as therapeutical approaches. We used Recombinant Bacterial Thermal Shift Assay to assess the binding of a substrate analog to phosphomannomutase-2 and the specific binding to phosphomannomutase-1 of an FDA-approved drug - clodronate. We also deepened the clodronate binding by enzyme activity assays and in silico docking. Our results confirmed the selective binding of clodronate to phosphomannomutase-1 and shed light on such binding.