Omniligase-1-mediated ligation for insulin analog synthesis in solu-tion and on phage surface

The B-chain C-terminal region of insulin has been mutated or modified to achieve improved therapeutic efficacies. For ex-ample, all FDA-approved insulin analogs have altered C-terminal segments, which leads to improved pharmacokinetic prop-erties and provide significant clinical benefits on blood sugar regulation. Nonetheless, there is still no efficient method to synthesize insulin analogs with the altered C-terminal region. Herein, we report a facile synthesis using omniligase-1 to li-gate an insulin core with a peptide segment in high conversion. We further apply this ligation to M13 phage surface modifi-cations and demonstrate that the phage displayed insulin molecules can bind to insulin receptor ectodomain in an insulin-dependent manner. These results pave the way for building phage display insulin library for therapeutic selections and demonstrate the feasibility of using omniligase-1 to display other disulfide-rich peptides and proteins on phage.