(427d) Engineered Self-Cleaving Purification Tags for Rapid Cloning and Protein Purification

Inteins are naturally-occurring protein elements that are capable of excising themselves from intein-containing precursor proteins. Inteins can be modified to cleave cleanly at their termini, and this function has been used to develop self-cleaving affinity tags for various applications. In particular, rational protein engineering and directed evolution was used to create the fast-cleaving ΔI-CM intein from the native Mycobacterium tuberculosis (Mtu) recA intein. This small, self-cleaving mutant intein is responsive to pH and temperature, and has been paired with a variety of conventional and novel purification tags to generate very convenient recombinant protein purification methods. In concurrent work in other laboratories, advances in molecular biology have made cloning techniques simpler and more efficient. Two technologies in this area, marketed by Invitrogen, use site-specific recombination to move genes into and between plasmids without conventional restriction and ligation steps. However, these methods require recombination sites to flank the target gene in the resulting vectors, which can complicate the use of self-cleaving intein tag technology. In this work, we have solved this problem by engineering the ΔI-CM intein DNA sequence to create functional new inteins that are compatible with the TOPO^® and Gateway^® cloning systems from Invitrogen. These new inteins include the required recombination sites, yet retain their pH and temperature-sensitive characteristics, cleaving at similar or slightly slower rates compared to the original ΔI-CM intein. Compatibility with the TOPO^® and Gateway^® expression systems allows rapid examination of various combinations of purification tag and expression host in initial process development for a given target. These modifications thus greatly increase the convenience of intein-based purification systems, and further their potential impact in a wide variety of biotechnological applications.