Elimination of in vivo cleavage between target protein and intein in the intein-mediated protein purification systems.

IMPACT is a novel protein purification system developed by New England BioLabs. This system utilizes the inducible self-cleavage activity of a protein splicing element (termed intein) to separate the target protein from the affinity intein-CBD tag without using any proteases. The IMPACT system has been widely used as an easy, one-step protein purification procedure. However, IMPACT usually produces a lower target protein yield than other protein purification systems, such as the pET system. It was found that the lower yield of the target protein, in many cases, was due to in vivo cleavage between the target protein and the intein during protein expression. Self-cleavage requires the first residue of the intein to be a cysteine with a free sulfhydryl group. In order to eliminate this in vivo auto-cleavage, we designed two single cysteine mutants in the intein protein to allow potential disulfide bond formation with the first cysteine. Therefore, no free sulfhydryl group in the first cysteine of the intein is available during protein expression. Our data indicated that the in vivo auto-cleavage during bacterial expression was completely eliminated with the intein mutation, resulting in a significant enhancement of the protein yield of the IMPACT expression system, comparable to the other high-level expression systems.