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(122c) Elastin-Like-Polypeptide And Polyhydroxybutyrate-Intein Mediated Protein Purification

Gillies, A., Princeton University
Wood, D. W., The Ohio State University

Novel alternatives to conventional affinity-tag separations have been developed for the purification of recombinant proteins. These systems use either a self-aggregating elastin-like polypeptide (ELP) tag or host-expressed polyhydroxybutyrate (PHB) granules with a PHB-binding tag on the protein. In both cases, the tags self-cleave following purification of the target, which is controlled by a small shift in pH. The ELP and PHB systems effectively replace the affinity resins and apparatus used in conventional systems, thus providing an inexpensive and simple non-chromatographic bioseparations technique. This method has been very successful in E. coli, yielding 35-120 milligrams of purified product per liter of liquid cell culture in shake flasks, depending on the target and system used. In our recent work, the ELP system has been used to purify a number of more unstable and aggregation-prone proteins, including organophosphate hydrolase. The system has also been used to purify the intact RNA polymerase holoenzyme by tagging a single subunit. This demonstrates the potential of this system to purify large multi-subunit protein complexes rapidly and inexpensively, without multiple columns. It could also be used to identify proteins that associate with a tagged target protein in protein-protein interaction fishing experiments. Further, the ELP and PHB purification systems are currently being combined with the GatewayTM cloning technology from Invitrogen using a GatewayTM-compatible intein. Destination vectors containing the ELP or PHB-binding phasin tagged-intein have been constructed, and can be used to create expression vectors containing the gene for the product protein of interest using conventional GatewayTM cloning. This will combine fast cloning with inexpensive purification, with the potential to allow rapid and cheap purification of proteins in a high-throughput manner.