Production of unnaturally linked chimeric proteins using a combination of sortase-catalyzed transpeptidation and click chemistry

Nat Protoc. 2013 Sep;8(9):1808-19. doi: 10.1038/nprot.2013.103. Epub 2013 Aug 29.


Chimeric proteins, including bispecific antibodies, are biological tools with therapeutic applications. Genetic fusion and ligation methods allow the creation of N-to-C and C-to-N fused recombinant proteins, but not unnaturally linked N-to-N and C-to-C fusion proteins. This protocol describes a simple procedure for the production of such chimeric proteins, starting from correctly folded proteins and readily available peptides. By equipping the N terminus or C terminus of the proteins of interest with a set of click handles using sortase A, followed by a strain-promoted click reaction, unnatural N-to-N and C-to-C linked (hetero) fusion proteins are established. Examples of proteins that have been conjugated via this method include interleukin-2, interferon-α, ubiquitin, antibodies and several single-domain antibodies. If the peptides, sortase A and the proteins of interest are in hand, the unnaturally N-to-N and C-to-C fused proteins can be obtained in 3-4 d.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminoacyltransferases / chemistry*
  • Bacterial Proteins / chemistry*
  • Carbon / chemistry
  • Chromatography, High Pressure Liquid
  • Click Chemistry / methods*
  • Cysteine Endopeptidases / chemistry*
  • Nitrogen / chemistry
  • Peptide Fragments / biosynthesis
  • Protein Engineering / methods*
  • Protein Folding
  • Recombinant Fusion Proteins / chemistry
  • Staphylococcus aureus / enzymology


  • Bacterial Proteins
  • Peptide Fragments
  • Recombinant Fusion Proteins
  • Carbon
  • Aminoacyltransferases
  • sortase A
  • Cysteine Endopeptidases
  • Nitrogen