Factor Xa recognition site

Four-residue site, IEGR, cleaved by activated coagulation factor Xa at the carboxyl side of the arginine. Placed between a tag and its cargo, it lets the N-terminal tag be removed after purification, and because the cut falls after the arginine it leaves no residue of the site on the cargo.

Length: 12 bp

Subtype: protease_site

Origin: Factor Xa recognition sequence IEGR

Characteristics

Encodes IEGR (4 amino acids, 12 nucleotides). Factor Xa cleaves at the carboxyl side of I-E[D]-G-R-X1, where X1 can be any residue except Arg and Pro, so the released cargo keeps its native N terminus. Cleavage runs at 4 to 25 °C. Factor Xa is a serine protease of the coagulation cascade, so the site is cut by a mammalian enzyme rather than a viral one such as TEV protease.

Applications: A removable junction between an N-terminal affinity tag and the protein of interest, cleaved with factor Xa after capture so that the tag is removed completely. Factor Xa has been less popular because cleavage needs longer incubation, but there are several examples of successful use.

Limitations: Factor Xa cleavage requires longer incubation time and is less effective. It has sometimes failed with insoluble fusion proteins or in denaturing reagents, and non-specific digestion has been reported. Five glycine residues next to the site can increase cleavage. It adds a protease step.

Sequence

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References

  1. K. Terpe (2003). Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol - Terpe 2003 Tag Fusions
  2. Bell et al. (2013). To fuse or not to fuse: what is your purpose?. Protein Sci - Bell 2013 Fusion Purpose