Enterokinase (enteropeptidase) recognition site
Five-residue site, DDDDK, cleaved by enterokinase (enteropeptidase) at the carboxyl side of the lysine. Placed between a tag and its cargo, it lets the tag be removed after purification, and because the cut falls after the lysine it leaves no residue of the site on the cargo.
Origin: Enterokinase recognition sequence (Asp)4-Lys
Characteristics
Encodes DDDDK (5 amino acids, 15 nucleotides). Enterokinase, also called enteropeptidase, recognizes D-D-D-D-K-X1 and cleaves at the carboxyl side of the lysine, so the released cargo keeps its native N terminus. Cleavage depends on residue X1. In a GST-calmodulin fusion digested for 16 h at 37 °C, 88% was cleaved with Ala at X1, 67% with Trp and 61% with Pro. The FLAG tag (DYKDDDDK) ends in this sequence, so enterokinase removes it.
Applications: A removable junction between an affinity or solubility tag and the protein of interest, cleaved with enterokinase after capture. Enterokinase is often the protease of choice for N-terminal fusions. The site also lets the FLAG tag be removed, because the C-terminal five residues of FLAG are this recognition sequence.
Limitations: Enterokinase shows sporadic cleavage at other residues at low levels, depending on the conformation of the protein substrate, and has been reported to cut at sites other than the engineered one. Cleavage falls with the residue after the lysine (61% with Pro, 88% with Ala). A FLAG tag in the same protein carries an internal site. It adds a protease step.
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