Thrombin recognition site
Six-residue site, LVPRGS, cleaved by thrombin between the arginine and the glycine. Placed between a tag and its cargo, it lets the tag be removed after purification, leaving a short glycine-serine remnant at the N terminus of the cargo.
Origin: Thrombin recognition sequence LVPRGS
Characteristics
Encodes LVPRGS (6 amino acids, 18 nucleotides). Thrombin cuts after the arginine in LVPR-GS, so the released cargo keeps two residues, GS, at its N terminus. The thrombin consensus is X4-X3-P-R[K]-X1'-X2', with hydrophobic X4 and X3 and non-acidic X1' and X2'. Cleavage runs at 20 to 37 °C for 0.3 to 16 h in Tris buffer at pH 8, where NaCl in the buffer inhibits.
Applications: A removable junction between an affinity or solubility tag and the protein of interest, cleaved with thrombin after capture. Thrombin is widely used to cleave tags, and LVPRGS is one of the frequently used recognition sites. LVPRGS also sits in the linker GGGGSLVPRGSGGGGS that tethers peptides to MHC class II chains in structural studies.
Limitations: Thrombin also recognizes short sites such as A-R-G and G-K-A, so a cargo with internal sites can be cut in the wrong place. Cleavage leaves a GS remnant on the cargo, unlike enterokinase and factor Xa, and it adds a protease step. Five glycine residues between the site and an N-terminal tag enhance cleavage.
Sequence
References
- K. Terpe (2003). Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol - Terpe 2003 Tag Fusions
- Bell et al. (2013). To fuse or not to fuse: what is your purpose?. Protein Sci - Bell 2013 Fusion Purpose
- Reddy Chichili et al. (2013). Linkers in the structural biology of protein–protein interactions. Protein Sci - ReddyChichili 2013 Linkers