title: Sortase-catalyzed transformations that improve the properties of cytokines.
abstract: Recombinant protein therapeutics often suffer from short circulating half-life and poor stability, necessitating multiple injections and resulting in limited shelf-life. Conjugation to polyethylene glycol chains (PEG) extends the circulatory half-life of many proteins, but the methods for attachment often lack specificity, resulting in loss of biological activity. Using four-helix bundle cytokines as an example, we present a general platform that uses sortase-mediated transpeptidation to facilitate site-specific attachment of PEG to extend cytokine half-life with full retention of biological activity. Covalently joining the N and C termini of proteins to obtain circular polypeptides, again executed using sortase, increases thermal stability. We combined both PEGylation and circularization by exploiting two distinct sortase enzymes and the use of a molecular suture that allows both site-specific PEGylation and covalent closure. The method developed is general, uses a set of easily accessible reagents, and should be applicable to a wide variety of proteins, provided that their termini are not involved in receptor binding or function.
authors.affiliation: Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA.
authors.forename: Maximilian W
authors.initials: MW
authors.lastname: Popp
authors.forename: Stephanie K
authors.initials: SK
authors.lastname: Dougan
authors.forename: Tzu-Ying
authors.initials: TY
authors.lastname: Chuang
authors.forename: Eric
authors.initials: E
authors.lastname: Spooner
authors.forename: Hidde L
authors.initials: HL
authors.lastname: Ploegh
mesh_terms: D019881:Aminoacyltransferases / Q000008:administration & dosage / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000008:administration & dosage / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D003500:Cyclization; D003546:Cysteine Endopeptidases / Q000008:administration & dosage / Q000737:chemistry* / Q000378:metabolism; D016207:Cytokines; D006207:Half-Life; D008722:Methods; D051379:Mice; D011092:Polyethylene Glycols / Q000737:chemistry*; D055550:Protein Stability; D013297:Streptococcus pyogenes
chemical_list: D001426:Bacterial Proteins; D016207:Cytokines; D011092:Polyethylene Glycols; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases