Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions
Methods for site-specific modification of proteins should be quantitative and versatile with respect to the nature and size of the biological or chemical targets involved. They should require minimal modification of the target, and the underlying reactions should be completed in a reasonable amount...
| 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/1721.1/106603 https://orcid.org/0000-0002-1090-6071 |
| _version_ | 1811087478146203648 |
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| author | Guimaraes, Carla P Witte, Martin D Theile, Christopher S Bozkurt, Gunes Kundrat, Lenka Blom, Annet E M Ploegh, Hidde |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Guimaraes, Carla P Witte, Martin D Theile, Christopher S Bozkurt, Gunes Kundrat, Lenka Blom, Annet E M Ploegh, Hidde |
| author_sort | Guimaraes, Carla P |
| collection | MIT |
| description | Methods for site-specific modification of proteins should be quantitative and versatile with respect to the nature and size of the biological or chemical targets involved. They should require minimal modification of the target, and the underlying reactions should be completed in a reasonable amount of time under physiological conditions. Sortase-mediated transpeptidation reactions meet these criteria and are compatible with other labeling methods. Here we describe the expression and purification conditions for two sortase A enzymes that have different recognition sequences. We also provide a protocol that allows the functionalization of any given protein at its C terminus, or, for select proteins, at an internal site. The target protein is engineered with a sortase-recognition motif (LPXTG) at the place where modification is desired. Upon recognition, sortase cleaves the protein between the threonine and glycine residues, facilitating the attachment of an exogenously added oligoglycine peptide modified with the functional group of choice (e.g., fluorophore, biotin, protein or lipid). Expression and purification of sortase takes ∼3 d, and sortase-mediated reactions take only a few minutes, but reaction times can be extended to increase yields. |
| first_indexed | 2024-09-23T13:46:43Z |
| format | Article |
| id | mit-1721.1/106603 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T13:46:43Z |
| publishDate | 2017 |
| publisher | Nature Publishing Group |
| record_format | dspace |
| spelling | mit-1721.1/1066032022-09-28T16:07:18Z Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions Guimaraes, Carla P Witte, Martin D Theile, Christopher S Bozkurt, Gunes Kundrat, Lenka Blom, Annet E M Ploegh, Hidde Massachusetts Institute of Technology. Department of Biology Whitehead Institute for Biomedical Research Ploegh, Hidde Methods for site-specific modification of proteins should be quantitative and versatile with respect to the nature and size of the biological or chemical targets involved. They should require minimal modification of the target, and the underlying reactions should be completed in a reasonable amount of time under physiological conditions. Sortase-mediated transpeptidation reactions meet these criteria and are compatible with other labeling methods. Here we describe the expression and purification conditions for two sortase A enzymes that have different recognition sequences. We also provide a protocol that allows the functionalization of any given protein at its C terminus, or, for select proteins, at an internal site. The target protein is engineered with a sortase-recognition motif (LPXTG) at the place where modification is desired. Upon recognition, sortase cleaves the protein between the threonine and glycine residues, facilitating the attachment of an exogenously added oligoglycine peptide modified with the functional group of choice (e.g., fluorophore, biotin, protein or lipid). Expression and purification of sortase takes ∼3 d, and sortase-mediated reactions take only a few minutes, but reaction times can be extended to increase yields. National Institutes of Health (U.S.) (Grant RO1 AI08787) 2017-01-24T20:24:32Z 2017-01-24T20:24:32Z 2013-08 Article http://purl.org/eprint/type/JournalArticle 1754-2189 1750-2799 http://hdl.handle.net/1721.1/106603 Guimaraes, Carla P et al. “Site-Specific C-Terminal and Internal Loop Labeling of Proteins Using Sortase-Mediated Reactions.” Nature Protocols 8.9 (2013): 1787–1799. https://orcid.org/0000-0002-1090-6071 en_US http://dx.doi.org/10.1038/nprot.2013.101 Nature Protocols Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf Nature Publishing Group PMC |
| spellingShingle | Guimaraes, Carla P Witte, Martin D Theile, Christopher S Bozkurt, Gunes Kundrat, Lenka Blom, Annet E M Ploegh, Hidde Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions |
| title | Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions |
| title_full | Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions |
| title_fullStr | Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions |
| title_full_unstemmed | Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions |
| title_short | Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions |
| title_sort | site specific c terminal and internal loop labeling of proteins using sortase mediated reactions |
| url | http://hdl.handle.net/1721.1/106603 https://orcid.org/0000-0002-1090-6071 |
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