Site-specific protein modification using immobilized sortase in batch and continuous-flow systems
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 describe...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | en_US |
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Nature Publishing Group
2017
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| Online Access: | http://hdl.handle.net/1721.1/109371 https://orcid.org/0000-0002-1090-6071 |
| _version_ | 1826188369438179328 |
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| author | 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. |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology 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. |
| author_sort | Witte, Martin D |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T07:58:35Z |
| format | Article |
| id | mit-1721.1/109371 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T07:58:35Z |
| publishDate | 2017 |
| publisher | Nature Publishing Group |
| record_format | dspace |
| spelling | mit-1721.1/1093712022-09-23T10:02:43Z Site-specific protein modification using immobilized sortase in batch and continuous-flow systems 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. Massachusetts Institute of Technology. Department of Biology Ploegh, Hidde 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. United States. National Institutes of Health (RO1 AI087879) 2017-05-26T14:48:49Z 2017-05-26T14:48:49Z 2015-02 Article http://purl.org/eprint/type/JournalArticle 1754-2189 1750-2799 http://hdl.handle.net/1721.1/109371 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 https://orcid.org/0000-0002-1090-6071 en_US http://dx.doi.org/10.1038/nprot.2015.026 Nature Protocols Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/ application/pdf Nature Publishing Group PMC |
| spellingShingle | 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. Site-specific protein modification using immobilized sortase in batch and continuous-flow systems |
| title | Site-specific protein modification using immobilized sortase in batch and continuous-flow systems |
| title_full | Site-specific protein modification using immobilized sortase in batch and continuous-flow systems |
| title_fullStr | Site-specific protein modification using immobilized sortase in batch and continuous-flow systems |
| title_full_unstemmed | Site-specific protein modification using immobilized sortase in batch and continuous-flow systems |
| title_short | Site-specific protein modification using immobilized sortase in batch and continuous-flow systems |
| title_sort | site specific protein modification using immobilized sortase in batch and continuous flow systems |
| url | http://hdl.handle.net/1721.1/109371 https://orcid.org/0000-0002-1090-6071 |
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