Hippo pathway towards cancer therapy: expression and purification of recombinant human Yap1 and Taz fragments in Escherichia coli

In Eukaryotes, the Hippo signaling pathway is an important regulator of cellular proliferation and organ size. The downstream effectors of the pathway are Yap1 and Taz, two cytoplasmic proteins which can enter the nucleus when they are not phosphorylated on critical aminoacidic residues, and the Tead transcription factors. The association of Yap1 or Taz with the Tead proteins allows the transcription of different genes involved in resistance to apoptosis and cell cycle progression. The Hippo pathway has been demonstrated to be crucial during embryo development, and its deregulation can play a role at different stages of many human cancers. Finding a way to modulate Hippo signaling could represent a promising step forward in in the field of cancer therapy but, on the other hand, also in the field of regenerative medicine. In this thesis work, we expressed recombinant fragments of both Yap1 and Taz proteins, with the purpose to achieve pure material to be used for X-ray crystallographic analysis. The final goal of this project was to define the three-dimensional structures of the proteins to provide new prospective for future pharmacological modulation of the pathway activity. Escherichia coli bacteria belonging to BL21(DE3) and SHuffle T7 strains were transformed with expression vectors encoding for the protein domains of interest either as single fragments or as recombinant variants fused with Maltose Binding Protein (MBP). The most stable fragments, Yap1 from amino acid 50 to 268 (Yap50-268) and Taz Tead Binding Domain (Taz TBD), have been purified through fast protein liquid chromatography with satisfactory yields and purity of the final material, used to set crystallographic screenings (Taz TBD). As a future step, the purified protein fragments would also represent an ideal substrate for protein-to-protein interaction studies with the Tead1 binding partner, in the aim of selecting pharmacological strategies to impair Yap1 and Taz activity in the context of cancer.