After identifying the optimized ligation condition, phage clone with FYTPKT at N-terminus of pIII was incubated with 20 μM omniligase-1 and 2 mM insulin ester 1f for 20 h at room temperature. After verifying the success of ligation by immunoblot with anti-hIns antibody (Figure ), the phage clones were purified by two rounds of PEG participation to remove excess 1f in the solution. pIII is critical to infect its bacterial host for amplification and this ability may be sabotaged with a modified pIII. We showed that omniligase-1mediated insulin ligation did not affect its viability (Figure ). To further investigate the biological function of ligated insulin on phage, 10 9 phage were incubated with magnetic beads, which were precoated with biotinylated IR ectodomain or biotin along. After rounds of wash to remove unbound phage by 0.1% PBST, the bound phage was eluted, and the yield of binding was determined by phage titration. The insulin-displaying phage had the highest yield (~20%) for positive binding signals, and all other control groups only had background signals (Figure ). We further performed a competitive binding assay using hIns as a soluble competitor. With increased concentrations of hIns, the percentage of phage binding to IR decreased, showing a dose-dependent IRspecific binding (Figure ). These results indicated that insulin ester 1f can be selectively ligated on phage pIII using omniligase-1. Furthermore, the displayed insulin can bind to its binding receptor in an insulin-dependent manner. Previously, we reported the use of a yeast display system to display singlechain insulin analogs with functional properties. However, a two-chain insulin cannot be achieved due to the complication of insulin C-peptide removal. To our best knowledge, this is the first demonstration of a two-chain insulin in a display system that demonstrated functional properties. This omniligase-1-mediated ligation also paves the way for displaying other disulfide-rich peptides or proteins on phage. We reported a synthetic method of insulin through the traceless omniligase-1-mediated ligation between a peptide (FYTPKT) as the B-chain C-terminal region and an estercontaining insulin core (1f) with a high conversion rate. The resulting ligated B21Ala hIns (2) had a comparable cell-based IR-stimulative signaling potency with the commercial recombinant hIns. The insulin ester 1f was able to be selectively conjugated to M13 phage pIII expressing sequence FYTPKT at the N-terminus by omniligase-1 without observable insulin-pVIII conjugates and with other phage proteins. The phage displayed insulin on pIII could bind to IR ectodomain in an insulin-dependent manner. To the best of our knowledge, this was the first demonstration of a two-chain insulin in a display system that presented functional properties. These results established a foundation of generating phage display insulin library for therapeutic selections and demonstrated the potential for omniligase-1 to display other disulfide-rich peptides and proteins on phage.