Sortase-catalyzed transformations that improve the properties of cytokines

Proc Natl Acad Sci U S A. 2011 Feb 22;108(8):3169-74. doi: 10.1073/pnas.1016863108. Epub 2011 Feb 4.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Aminoacyltransferases / administration & dosage
  • Aminoacyltransferases / chemistry*
  • Aminoacyltransferases / metabolism
  • Animals
  • Bacterial Proteins / administration & dosage
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / metabolism
  • Catalysis
  • Cyclization
  • Cysteine Endopeptidases / administration & dosage
  • Cysteine Endopeptidases / chemistry*
  • Cysteine Endopeptidases / metabolism
  • Cytokines
  • Half-Life
  • Methods
  • Mice
  • Polyethylene Glycols / chemistry*
  • Protein Stability*
  • Streptococcus pyogenes

Substances

  • Bacterial Proteins
  • Cytokines
  • Polyethylene Glycols
  • Aminoacyltransferases
  • sortase A
  • Cysteine Endopeptidases