Abstract
Therapeutic proteins are indispensable in treating numerous human diseases. However, therapeutic proteins often suffer short serum half-life. In order to extend the serum half-life, a natural albumin ligand (a fatty acid) has been conjugated to small therapeutic peptides resulting in a prolonged serum half-life via binding to patients' serum albumin in vivo. However, fatty acid-conjugation has limited applicability due to lack of site-specificity resulting in the heterogeneity of conjugated proteins and a significant loss in pharmaceutical activity. In order to address these issues, we exploited the site-specific fatty acid-conjugation to a permissive site of a protein, using copper-catalyzed alkyne-azide cycloaddition, by linking a fatty acid derivative to p-ethynylphenylalanine incorporated into a protein using an engineered pair of yeast tRNA/aminoacyl tRNA synthetase. As a proof-of-concept, we show that single palmitic acid conjugated to superfolder green fluorescent protein (sfGFP) in a site-specific manner enhanced a protein's albumin-binding in vitro about 20 times and the serum half-life in vivo 5 times when compared to those of the unmodified sfGFP. Furthermore, the fatty acid conjugation did not cause a significant reduction in the fluorescence of sfGFP. Therefore, these results clearly indicate that the site-specific fatty acid-conjugation is a very promising strategy to prolong protein serum half-life in vivo without compromising its folded structure and activity.