| Biotin–aminohexanoic acid–LPETGS–NH₂ is a synthetic peptide used extensively in sortase A–mediated transpeptidation assays and proximity-based cell interaction studies, notably in the LIPSTIC (Labeling Immune Partnerships by SorTagging Intercellular Contacts) methodology.
The LPETGS motif belongs to the LPXTG sequence family recognized by Sortase A (SrtA) from Staphylococcus aureus—a membrane-associated enzyme that cleaves specifically between the threonine and glycine residues in the LPXTG motif. This reaction yields a reactive thioester intermediate that can be resolved by attack from an oligoglycine nucleophile, enabling site-specific ligation of biomolecules.
Applications:
1. Cell-Cell Interaction Mapping (LIPSTIC)
- In the LIPSTIC system, biotin-Ahx-LPETGS serves as the donor substrate: cells engineered to express Sortase A on their surface transfer this biotinized peptide to adjacent cells expressing an oligoglycine acceptor tag fused to a receptor or ligand.
- This strategy allows direct marking of contacting cells, which can later be recovered and analyzed via flow cytometry or immunoprecipitation.
2. Protein Engineering and Labeling
- Separate from LIPSTIC, LPETG-based peptides (including LPETGS) are widely used to site-specifically tag proteins or antibodies with biotin, fluorescent dyes, or other probes using sortase-mediated ligation.
Advantages of This Peptide Design:
- Biotin modification provides an exceptionally strong, non-covalent affinity to streptavidin or neutravidin, facilitating highly sensitive detection or streptavidin-based pull-down assays.
- The Ahx spacer (6‑aminohexanoic acid) ensures that the biotin tag is sufficiently distanced from the LPETGS core motif, reducing steric hindrance during enzymatic conjugation or receptor binding.
- The terminal amide (–NH₂) stabilizes the C-terminus to prevent carboxylate interference in enzymatic processing or charge-based artifacts.
- Mechanistic studies of immune cell interactions: LIPSTIC enables researchers to capture and quantify receptor–ligand encounters between living cells in vivo, shedding light on dynamic immune synapses.
- Versatility across biological settings: The peptide is adaptable to diverse receptor-ligand systems as long as one partner is engineered to express Sortase A and another carries glycine residues that function as nucleophiles.
- Precision and specificity: Sortase-based ligation produces site-specific modifications without general chemical reactivity, lowering background labeling and enhancing reproducibility.
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4. Pasqual, G., Chudnovskiy, A., Tas, J. et al. Monitoring T cell–dendritic cell interactions in vivo by intercellular enzymatic labelling. Nature 553, 496–500 (2018). https://doi.org/10.1038/nature25442
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