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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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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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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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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DOI: https://doi.org/10.1038/nprot.2015.026
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