Unexpected tobacco etch virus (TEV) protease cleavage of recombinant human proteins

The tobacco etch virus (TEV) protease is a commonly used reagent for removal of solubility and purification tags from recombinant proteins and is cited as being highly specific for its canonical cleavage site. Flexibility in some amino acids within this recognition sequence has been described in the literature but researchers generally assume few native human proteins will carry off-target sequences for TEV cleavage. We report here the aberrant cleavage of three human proteins with non-canonical TEV protease cleavage sites and identify broader sequence specificity rules that can be used to predict unwanted cleavage of recombinant proteins. Using these rules, 456 human proteins were identified that could be substrates for unwanted TEV protease cleavage.