Site-specific protein modification using immobilized sortase in batch and continuous-flow systems

Martin D. Witte, Tongfei Wu, Carla P. Guimaraes, Christopher S. Theile, Annet E. M. Blom, Jessica R. Ingram, Zeyang Li, Lenka Kundrat, Shalom D. Goldberg, Hidde L. Ploegh*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

40 Citations (Scopus)

Abstract

Transpeptidation catalyzed by sortase A allows the preparation of proteins that are site-specifically and homogeneously modified with a wide variety of functional groups, such as fluorophores, PEG moieties, lipids, glycans, bio-orthogonal reactive groups and affinity handles. This protocol describes immobilization of sortase A on a solid support (Sepharose beads). Immobilization of sortase A simplifies downstream purification of a protein of interest after labeling of its N or C terminus. Smaller batch and larger-scale continuous-flow reactions require only a limited amount of enzyme. The immobilized enzyme can be reused for multiple cycles of protein modification reactions. The described protocol also works with a Ca2+-independent variant of sortase A with increased catalytic activity. This heptamutant variant of sortase A (7M) was generated by combining previously published mutations, and this immobilized enzyme can be used for the modification of calcium-senstive substrates or in instances in which low temperatures are needed. Preparation of immobilized sortase A takes 1-2 d. Batch reactions take 3-12 h and flow reactions proceed at 0.5 ml h(-1), depending on the geometry of the reactor used.

Original languageEnglish
Pages (from-to)508-516
Number of pages9
JournalNature protocols
Volume10
Issue number3
DOIs
Publication statusPublished - Mar-2015
Externally publishedYes

Keywords

  • STAPHYLOCOCCUS-AUREUS
  • MEDIATED REACTIONS
  • LIGATION
  • PEPTIDE
  • DISPLAY
  • LOOP

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