Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions
Author(s)
Guimaraes, Carla P; Witte, Martin D; Theile, Christopher S; Bozkurt, Gunes; Kundrat, Lenka; Blom, Annet E M; Ploegh, Hidde; ... Show more Show less
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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.
Date issued
2013-08Department
Massachusetts Institute of Technology. Department of Biology; Whitehead Institute for Biomedical ResearchJournal
Nature Protocols
Publisher
Nature Publishing Group
Citation
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.
Version: Author's final manuscript
ISSN
1754-2189
1750-2799