Sortase-catalyzed transformations that improve the properties of cytokines

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 108; no. 8; pp. 3169 - 3174
• Main Authors: Popp, Maximilian W, Dougan, Stephanie K, Chuang, Tzu-Ying, Spooner, Eric, Ploegh, Hidde L
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 22.02.2011; National Acad Sciences
• Subjects: Aminoacyltransferases - administration & dosage; Aminoacyltransferases - chemistry; Aminoacyltransferases - metabolism; Animals; Bacterial Proteins - administration & dosage; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Biological Sciences; Catalysis; Cell growth; Cell lines; Chromatography; Cyclization; Cysteine Endopeptidases - administration & dosage; Cysteine Endopeptidases - chemistry; Cysteine Endopeptidases - metabolism; Cytokines; Enzymes; Epics; Half lives; Half-Life; Methods; Mice; Nucleophiles; Polyethylene glycol; Polyethylene Glycols - chemistry; Polypeptides; Protein Stability; Proteins; Receptor mechanisms; Receptors; Shelf life; sortase; Streptococcus pyogenes; Sutures; Thermal stability; Transformation
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1016863108

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.