Overabundant anti-apoptotic proteins, such as Bcl-x_L, form heterodimers with pro-apoptotic proteins, such as Bak, to prevent the pro-apoptotic proteins from properly functioning, thereby causing cancer. We are attempting to design variants of Bak that will bind tightly with the overabundant anti-apoptotic protein Bcl-x_L in certain cancer cells, thus acting as a competitive inhibitor of the binding with the pro-apoptotic protein Bak. As a first step towards this goal, we have recapitulated the binding of Bak BH3 peptide and Bcl-x_Lon the E. coli. cell surface. We also diplayed Bak peptides of different lengths and regions to study their binding affinity towards Bcl-x_L. Experiments show that use of eCPX display system leads to successful display of Bak peptides as judged by Western blotting. The recent binding assay between Bak BH3 peptide and fluorescent Bcl-x_L using flow cytometry shows more than ten-fold increase over the negative control. We have also successfully displayed full length Bak protein using several E. coli. surface display systems, including Lpp-OmpA, APEx, ice nucleation protein and pgsA, and evaluated them for use in future directed evolution experiments to identify Bak variants with improved affinity toward Bcl-x_L.

Extended Abstract Status: Not Uploaded