FHL2 is an adapter protein that contains four and a half LIM domains, which mediate interactions with other proteins. Between LIM domains there are intrinsically disordered regions that enable bonding to broad range of different proteins. This is the reason why FHL2 is a part of many signalling pathways. For example: it plays an important role in cell signalling mediated by Epithelial Cell Adhesion Molecule (EpCAM), which is highly expressed in cancer cells. FHL2, as an adaptor protein, mediates assembly of multiprotein complex EpIC-FHL2-β-catenin that is formed in cytoplasm and transported into the nucleus, where it binds to transcriptional factor Lef-1 and promotes transcription of oncogenes that cause cell proliferation. It is not clear how the complex EpIC-FHL2-β-catenin assembles and its structure is also unkown.
The goal of our research was to optimize expression of recombinant FHL2 and to prepare sufficient amount for characterization of structural and biochemical properties of EpIC-FHL2-β-catenin complex. After induction of FHL2 expression in bacterial cells it is found in an insoluble fraction and it needs to be isolated from inclusion bodies. We carried out three experiments trying to recover recombinant FHL2 from inclusion bodies and we estimated the success of isolation for each one of them.
In all three cases, we observed the largest loss of FHL2 due to unsuccessful solubilization of inclusion bodies, where our protein remained in the form of soluble aggregates, despite the presence of denaturant. We observed the most promising results when we add non-ionic detergent Triton X-100, that prevents formation of agreggates, to the process of inclusion bodies dissolution in 8 M urea. Even though we were able to recover substantial amount of fusion protein, we observed its unstability and degradation during the process of dialysis and proteolysis with TEV protease. We concluded that the isolation of recombinant FHL2 from inclusion bodies is pointless and is thus more suitable to preform coexpression of FHL2 with its binding partner. This method is also in line with the role of FHL2 protein in a cell, where it is present as an adapter protein and is therefore always present in a multiprotein complex. On top of that it also contains disordered regions between LIM domains, which is why it is prone to aggregation.
To continue our work we would need to preform coexpression from one plasmid that would code for both proteins, FHL2 and β-catenin, each one with its own T7 promoter. If we carry out coexpression of FHL2 and β-catenin from different vectors, we get unequal expression of both proteins. With stable FHL2-β-catenin complex it would be possible to study interaction and structural properties of whole EpIC-FHL2-β-catenin complex.