The effectiveness of protein and peptide pharmaceuticals depends essentially on their intrinsic pharmacokinetics. Small-sized pharmaceuticals in particular often suffer from short serum half-lives due to rapid renal clearance. To improve the pharmacokinetics by association with serum albumin (SA) in vivo, we generated an SA-binding tag of a helix-loop-helix (HLH) peptide to be linked with protein pharmaceuticals. For use in future preclinical studies, screening of yeast-displayed HLH peptide libraries against human SA (HSA) and mouse SA (MSA) was alternately repeated to give the SA-binding peptide AY-VE, which exhibited cross-binding activities to HSA and MSA with KD of 65 and 20 nM, respectively. As a proof of concept, we site-specifically conjugated peptide AY-VE with insulin to examine its bioactivity in vivo. In mouse bioassay monitoring the blood glucose level, the AY-VE conjugate was found to have a prolonged hypoglycemic effect for 12 h. The HLH peptide tag is a general platform for extending the bioactivity of therapeutic peptides or proteins.
Keywords: CuAAC; helix−loop−helix; insulin; serum albumin; yeast surface display.