Omniligase-1-mediated insulin display on pIII. To further explore omniligase-1-mediated pIII labeling, phage clone with FYTPKT displayed at the N-terminus of pIII was incubated with different concentrations of insulin ester 1f and omniligase-1 for 20 h at 37 o C or room temperature (Figure ). The reaction was monitored by immunoblot using anti-pIII antibody and anti-hIns antibody. From the blots (Figure ), a new pIII band was detected around ~65 kDa, corresponding to the insulin-pIII fusion product. We found the ligation efficiency was higher at room temperature than 37 o C. The lower amount of insulin-pIII adduct in 37 o C indicated that pIII was proteolyzed by omniligase-1 due to the protease activity of the enzyme. We further evaluated the reactions at even lower temperatures (4 o C and 12 o C) (Figure ) and found that room temperature led to the highest ligation efficiency with 20 μM omniligase-1 and 2 mM insulin ester 1f in which ~50% pIII was successfully ligated with insulin. A higher concentration of omniligase-1 (80 μM) did not generate even higher efficiency.