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

Nature Protocols volume 10, pages 508–516 (2015)Cite this article

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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.

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Figure 1: Comparison of wild-type (WT), pentamutant (5M) and heptamutant (7M) Srt A activity in the presence or absence of Ca2+.
Figure 2: Schematic representation of the sortase immobilization on Sepharose beads.
Figure 3: Comparison of C-terminal-specific labeling of an LPETG-containing streptavidin protein with a Gly3-TAMRA fluorophore using immobilized heptamutant (7M) sortase A (batch) or heptamutant sortase A in solution.
Figure 4: Comparison of N-terminal-specific labeling of a Gly3-containing cholera toxin B (CtxB) with an LPETG-containing TAMRA fluorophore using immobilized sortase A (batch) or sortase A in solution.
Figure 5: C-terminal labeling of proteins using immobilized sortase.
Figure 6: N-terminal labeling of proteins using immobilized sortase.

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Acknowledgements

This work was supported by funding from The Netherlands Organisation for Scientific Research (to M.D.W.) and the US National Institutes of Health (NIH; grant no. RO1 AI087879 to H.L.P.).

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Author notes

  1. Martin D Witte

    Present address: Present address: Bio-Organic Chemistry, Stratingh Institute for Chemistry, University of Groningen, Groningen, the Netherlands.,

  2. Martin D Witte, Tongfei Wu, Carla P Guimaraes and Christopher S Theile: These authors contributed equally to this work.

Authors and Affiliations

  1. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.,

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

  2. Oncology Medicinal Chemistry, Janssen Research and Development, Beerse, Belgium

    Tongfei Wu

  3. Centyrex, Janssen Research and Development, LLC, Spring House, Pennsylvania, USA

    Shalom D Goldberg

  4. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.,

    Hidde L Ploegh

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  1. Martin D Witte
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Contributions

M.D.W., C.P.G. and H.L.P. conceived of the idea; M.D.W., T.W., C.S.T., C.P.G. and A.E.M.B. performed the immobilization reactions and optimized the sortase reactions in batch and in flow; J.R.I. developed the heptamutant sortase; and Z.L. and L.K. assisted in sortase and protein production; M.D.W., T.W., C.S.T., C.P.G., J.R.I. and H.L.P. wrote the manuscript; and S.D.G. and H.L.P. supervised the project.

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Correspondence to Hidde L Ploegh.

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Witte, M., Wu, T., Guimaraes, C. et al. Site-specific protein modification using immobilized sortase in batch and continuous-flow systems. Nat Protoc 10, 508–516 (2015). https://doi.org/10.1038/nprot.2015.026

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