Site-specific protein modification using immobilized sortase in batch and continuous-flow systems
Author(s)
Witte, Martin D; Wu, Tongfei; Guimaraes, Carla P; Theile, Christopher S; Blom, Annet E M; Ingram, Jessica R; Kundrat, Lenka; Goldberg, Shalom D; Ploegh, Hidde; Li, Zeyang,S.M.Massachusetts Institute of Technology.; ... Show more Show less
DownloadPloegh_Site-specific.pdf (1.045Mb)
OPEN_ACCESS_POLICY
Open Access Policy
Creative Commons Attribution-Noncommercial-Share Alike
Terms of use
Metadata
Show full item recordAbstract
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.
Date issued
2015-02Department
Massachusetts Institute of Technology. Department of BiologyJournal
Nature Protocols
Publisher
Nature Publishing Group
Citation
Witte, Martin D; Wu, Tongfei; Guimaraes, Carla P; Theile, Christopher S; Blom, Annet E M; Ingram, Jessica R; Li, Zeyang; Kundrat, Lenka; Goldberg, Shalom D and Ploegh, Hidde L. “Site-Specific Protein Modification Using Immobilized Sortase in Batch and Continuous-Flow Systems.” Nature Protocols 10, no. 3 (February 2015): 508–516 © 2015 Macmillan Publishers Limited, part of Springer Nature
Version: Author's final manuscript
ISSN
1754-2189
1750-2799