In this work, our previously reported Delta I-CM and Delta I-G-CM mutant inteins were rationally re-engineered to be compatible with Invitrogen's Topo (R) 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 (R) cloning method. Addition of the Topo (R) 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 degrees C. In particular, the modified Delta I-GT-CM intein is compatible with both the Topo (R) and Gateway (R) 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. (C) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 26: 1205-1212, 2010