ABSTRACT:We have combined Invitrogen's Gateway 1 cloning technology with self-cleaving purification tags to generate a new system for rapid production of recombinant protein products. To accomplish this, we engineered our previously reported DI-CM cleaving intein to include a Gateway cloning recognition sequence, and demonstrated that the resulting Gateway-competent intein is unaffected. This intein can therefore be used in several previously reported purification methods, while at the same time being compatible with Gateway cloning. We have incorporated this intein into a set of Gateway vectors, which include selfcleaving elastin-like polypeptide (ELP), chitin binding domain (CBD), phasin (polyhydroxybutyrate-binding), or maltose binding domain (MBD) tags. These vectors were verified by Gateway cloning of TEM-1 b-lactamase and Escherichia coli catalase genes, and the expressed target proteins were purified using the four methods encoded on the vectors. The purification methods were unaffected by replacing the DI-CM intein with the Gateway intein. It was observed that some purification methods were more appropriate for each target than others, suggesting utility of this technology for rapid process identification and optimization. The modular design of the Gateway system and intein purification method suggests that any tag and promoter can be trivially added to this system for the development of additional expression vectors. This technology could greatly facilitate process optimization, allowing several targets and methods to be tested in a high-throughput manner.

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.