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; Li, Zeyang; Kundrat, Lenka; Goldberg, Shalom D; Ploegh, Hidde; ... Show more Show less
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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 Ca[superscript 2+]-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[superscript −1], depending on the geometry of the reactor used.
DepartmentMassachusetts Institute of Technology. Department of Biology; Whitehead Institute for Biomedical Research
Witte, Martin D., Tongfei Wu, Carla P. Guimaraes, Christopher S. Theile, Annet E. M. Blom, Jessica R. Ingram, Zeyang Li, Lenka Kundrat, Shalom D. Goldberg, and Hidde L. Ploegh. “Site-Specific Protein Modification Using Immobilized Sortase in Batch and Continuous-Flow Systems.” Nature Protocols 10, no. 3 (February 26, 2015): 508–516.
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