Site-specific protein modification using immobilized sortase in batch and continuous-flow systems
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Ploegh_Site-specific.pdf
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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.
Date Issued
February 2015
Journal
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
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.
MIT Department
Massachusetts Institute of Technology. Department of Biology
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DOI of Published Version
https://doi.org/10.1038/nprot.2015.026
