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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    • "Sortase-mediated ligation also utilizes small tag consisting of a few amino acids. However, this system requires a large excess of fluorophore ligand for efficient conversion , making it most useful for in vitro labeling applications where the conjugate can be purified (Theile et al., 2013). Collectively, improving the delivery of the dye labels to cells and tissue, decreasing the size of the genetically encoded tag, and expanding the compatibility with different fluorophores will open new avenues for single-molecule experiments in live cells. "
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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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