The tuberous sclerosis protein complex (pTSC) is an intracellular stress-sensor that nucleates a proteinaceous signaling hub to integrate regulatory information about the growth and energy consumption of the cell. The structure and stoichiometry of pTSC have been revealed through previous biochemical and cryo-electron microscopy (cryo-EM) studies, hinting at the possibility of pTSC forming large higher-order oligomers. However, these studies required the stabilization of the protein with chemical cross-linking or graphene oxide-coated grids during sample preparation, making it difficult to obtain a complete understanding of its architecture.
Though there has been immense progress in filling information gaps, there is still much to be learned with regards to understudied and/or unstable proteins and protein complexes, like pTSC. One of the major challenges that researchers often face is the effort to express, purify, and isolate proteins for downstream structural characterization. Purification and expression of recombinant proteins remain the preferred method for obtaining protein samples; however, this can be a demanding task for certain proteins.
Protein complexes may form higher-order structures only when they are expressed under native conditions within the host organism. Therefore, novel techniques for isolating proteins from their native sources allow for progress in characterizing those unstable, ‘tricky’ proteins. Next generation protein sequencing (NGPS) is one approach that is being leveraged in protein purification strategies. With NGPS, the primary amino acid sequences of commercially available mAbs are now being derived and utilized to engineer recombinant antibodies and antibody fragments that show improved target specificity for capturing proteins and protein complexes from their native sources. Strategies like these can help provide the structural and functional information towards our understanding of diverse proteins and their intricacies.