(219a) Development and Impacts of Intein-Based Bioseparations

Discovered in the early 1990â??s, inteins are self-slicing proteins with the ability to excise themselves from a variety of host proteins. This self-splicing activity allows inteins to be moved into many contexts for a range of applications, and mutated inteins have now been demonstrated in a wide variety of protein engineering, labeling and purification approaches. One of the most impactful of the intein applications has been the development of self-cleaving affinity tags for recombinant protein purification. In this strategy, a conventional affinity or purification tag is appended to a self-cleaving mutant intein. The intein is further appended to a target protein, which can be easily purified via the tag. Once purified, the intein is induced to self-cleave, releasing the purified native target with minimal expense or additional processing. Early intein applications were plagued by issues with premature cleaving, required modifications to the target protein, or the need for high concentrations of thiol compounds to induce cleaving. These issues limited the applications where inteins could be used successfully, and prevented inteins from gaining widespread adoption in scientific research and industrial protein production. Recent work has solved many of these problems, however, most notably allowing inteins to be used for disulfide-bonded recombinant glycoproteins expressed in mammalian and other eukaryotic hosts. Here we will report a split intein system developed in our laboratory, where a small intein-segment tag on the target protein is captured by novel affinity resin. Controlled self-cleavage of the smaller segment releases the native protein, without the need for modification of its sequence or additional processing. This approach has been highly successful in solving the remaining problems with inteins, and has potential as a platform for the purification of virtually any recombinant protein expressed in any host system.