Site-specific N-terminal labeling of proteins using sortase-mediated reactions

Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
Nature Protocol (Impact Factor: 9.67). 09/2013; 8(9):1800-7. DOI: 10.1038/nprot.2013.102
Source: PubMed


This protocol describes the use of sortase-mediated reactions to label the N terminus of any given protein of interest. The sortase recognition sequence, LPXTG (for Streptococcus aureus sortase A) or LPXTA (for Staphylococcus pyogenes sortase A), can be appended to a variety of probes such as fluorophores, biotin or even to other proteins. The protein to be labeled acts as a nucleophile by attacking the intermediate formed between the probe containing the LPXTG/A motif and the sortase enzyme. If sortase, the protein of interest and a suitably functionalized label are available, the reactions usually require less than 3 h.

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Available from: Hidde L Ploegh, Dec 08, 2014
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    • "GGG.TAMRA, AAA.AF647, TAMRA.LPETGG and Biotin.LPETGG were synthesized as described in [20] [21]. Soluble sortase labeling reactions with SrtAstrep and SrtAstaph were performed as described [19] [20] [21] [26]. The SrtAstaph7M has increased activity and reactions took place at 4°C and with 20% of sortase in relation to proaerolysin. "
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