Creacio, produccio i purificacio de noves variants de prolina de l’H6-ΔProΔLeuApoptina

Abstract

Cancer is a heterogeneous group of diseases that occur for the appearance of an uncontrolled proliferation of abnormal cells with the ability to invade other tissues in a process called metastasis. Metastasis occurs when genetically unstable cancer cells adapt to a tissue microenvironment that is distant from the primary tumor in a selection process. Currently, cancer causes high mortality worldwide and it has become an issue of concern for the scientific community. Current therapies, in most cases, have not presented the expected selectivity nor efficacy, so the research of anti-tumor drugs is based on new strategies. One of these strategies is the use of antitumor viral proteins, such as Apoptin, which is an intrinsically disordered protein from chicken anaemia virus (CAV) that has the ability to cause the death of tumor cells leaving the non-tumorous cells undamaged. This work is a contribution to the project initiated some years ago by the group of Protein Engineering of the Department of Biology of the University of Girona whose objective is the study of the mechanism that uses this protein to affect specifically the tumor cells. In particular, the role of the interaction of other proteins such as the cyclophilin Ppil3. Different variants of Apoptin (H6-ProLeuApoptina) have been created, produced and purified. In each variant, every residue of proline has been substituted by alanine to eliminate possible interaction targets for the protein Ppil3. The detected changes in this interaction could determine the key aminoacids that confers to Apoptin its cytotoxic effect. The introduction of these mutations has changed the behaviour of recombinant proteins during their purification and the initial protocol has been modified in order to obtain enough quantities to use them in further experiments. The main modification has been the use of the refolding by dialysis method which has demonstrated a significantly increasing of protein yield. Finally, when the different proteins have been successfully obtained, the interaction between the different variants of H6- ProLeuApoptina and Ppil3 was tried to be analysed by pull-down experiments