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|a dc
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|a Witte, Martin D
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|a Massachusetts Institute of Technology. Department of Biology
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|a Ploegh, Hidde
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|a Wu, Tongfei
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|a Guimaraes, Carla P
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|a Theile, Christopher S
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|a Blom, Annet E M
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|a Ingram, Jessica R
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|a Kundrat, Lenka
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|a Goldberg, Shalom D
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|a Ploegh, Hidde
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|a Li, Zeyang,S.M.Massachusetts Institute of Technology.
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|a Site-specific protein modification using immobilized sortase in batch and continuous-flow systems
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|b Nature Publishing Group,
|c 2017-05-26T14:48:49Z.
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|z Get fulltext
|u http://hdl.handle.net/1721.1/109371
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|a 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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|a United States. National Institutes of Health (RO1 AI087879)
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|a en_US
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|a Article
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|t Nature Protocols
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