Cleavage sites used in recombinant protein expression and purification

Published in reference R.C. Stevens. "The cost and value of three-dimensional protein structure" Drug Discovery World, 4, 35-48 (2003).

Excision site ↓ Cleavage Enzyme / Self-Cleavage Comments
Asp-Asp-Asp-Asp-Lys↓  Enterokinase *The site will not cleave if followed by a proline residue.
Secondary cleavage sites at other basic residues, depending on conformation of protein substrate. Active from pH 4.5 to 9.5 and between 4°C and 45°C.
Ile-Glu/Asp-Gly-Arg↓  Factor Xa protease Will not cleave if followed by proline and arginine. Secondary cleavage sites following Gly-Arg sequences.
Leu-Val-Pro-Arg↓Gly-Ser  Thrombin Secondary cleavage sites. Biotinylated form available for removal with immobilized streptavidin.
Glu-Asn-Leu-Tyr-Phe-Gln↓Gly  TEV protease Seven-residue recognition site, making it a highly site-specific protease. Active over a wide range of temperatures. Protease available as a His-tag fusion, allowing for protease removal after recombinant protein cleavage.
Leu-Glu-Val-Leu-Phe-Gln↓Gly-Pro  PreScission™ protease Genetically engineered form of human rhinovirus 3C protease with a GST fusion, allowing for facile cleavage and purification of GST-tagged proteins along with protease removal after recombinant protein cleavage. Enables low-temperature cleavage of fusion proteins containing the eight-residue recognition sequence.
Specific intein-encoded sequences Intein 1 & intein 2 Uses self-cleavable affinity tags. Even after cleavage unnatural termini are present on the protein of interest.
Signal sequences Signal peptidases Cleavage of leader sequence concomitant with protein export from the cytoplasm.
*LaVallie, E.R., McCoy, J.M., Smith, D.B. & Riggs, P. (1994). Enzymatic and chemical cleavage of fusion proteins. In Current Protocols in Molecular Biology. pp. 16.4.5-16.4.17, John Wiley and Sons, Inc, New York, NY.

 

last updated December 18, 2003 - send corrections and comments to Angela Walker (alwalker@scripps.edu)