Self-cleaving purification tags re-engineered for rapid Topo® cloning.

In this work, our previously reported ΔI-CM and ΔI(G)-CM mutant inteins were rationally re-engineered to be compatible with Invitrogen's Topo® cloning system. The resulting new inteins include the vaccinia virus topoisomerase I DNA recognition sequence TCCTT at their 3' ends, making them compatible with the highly convenient one-step Topo® cloning method. Addition of the Topo® recognition sequence resulted in an altered amino acid sequence at the C-termini of the inteins, changing their final five residues from VVVHN to VLVHN. Despite this change, these modified inteins retained their self-cleaving function, and continue to exhibit pH and temperature-sensitive cleaving characteristics as required for their use in generating self-cleaving affinity tags. Although the C-terminal modification decreased the intein cleavage rate under optimal conditions, cleavage can typically be completed within several hours at pH 6.5 and 37°C. In particular, the modified ΔI(GT)-CM intein is compatible with both the Topo® and Gateway® methods simultaneously, allowing fast parallel construction of multiple expression vectors with varying combinations of target proteins, self-cleaving affinity tags and promoters. These newly engineered inteins increase the functionality of intein-mediated technology, making it possible to explore a large number of combinations between target genes, self-cleaving affinity tags and expression hosts in a fast and efficient manner.