title: Site-specific fatty acid-conjugation to prolong protein half-life in vivo.
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.
authors.affiliation: Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22904, USA.
authors.forename: Sung In
authors.initials: SI
authors.lastname: Lim
authors.forename: Yukina
authors.initials: Y
authors.lastname: Mizuta
authors.forename: Akinori
authors.initials: A
authors.lastname: Takasu
authors.forename: Young S
authors.initials: YS
authors.lastname: Hahn
authors.forename: Yong Hwan
authors.initials: YH
authors.lastname: Kim
authors.forename: Inchan
authors.initials: I
authors.lastname: Kwon
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives* / Q000737:chemistry; D001386:Azides / Q000737:chemistry*; D002384:Catalysis; D003300:Copper / Q000737:chemistry; D061565:Cycloaddition Reaction; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D019308:Palmitic Acid / Q000737:chemistry*; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry
chemical_list: D001386:Azides; C408243:p-ethynylphenylalanine; D049452:Green Fluorescent Proteins; D019308:Palmitic Acid; D003300:Copper; D013762:Tetrahydrofolate Dehydrogenase; D000409:Alanine
keywords: Albumin; Copper-catalyzed alkyne-azide cycloaddition; Fatty acid; Half-life; Noncanonical amino acid