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

Martin D. Witte, Christopher S. Theile, Tongfei Wu, Carla P. Guimaraes, Annet E. M. Blom, Hidde L. Ploegh*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

57 Citations (Scopus)

Abstract

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-alpha, 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.

Original languageEnglish
Pages (from-to)1808-1819
Number of pages12
JournalNature protocols
Volume8
Issue number9
DOIs
Publication statusPublished - Sep-2013
Externally publishedYes

Keywords

  • AZIDE-ALKYNE CYCLOADDITION
  • BISPECIFIC ANTIBODIES
  • TERMINAL ALKYNES
  • LIGATION
  • SPECIFICITY
  • FUSIONS
  • CELLS
  • TAG

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