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(326a) Simple and Economical Purification of Recombinant Proteins by Self-Cleaving Aggregation Tags

Wood, D. W., The Ohio State University
Chu, H., Princeton University
Mee, C., Princeton University
Banki, M. R., Princeton University

Two simple and general methods for the purification of recombinant protein products expressed in Escherichia coli are reported. These methods provide a pure, native target protein without the need for chromatography or affinity separations of any kind. Each involves a fusion of a given target-protein gene to one of two self-cleaving aggregation tags. The first tag is a reversibly precipitating elastin-like protein (ELP). Gentle heating causes the tag to self-assemble and fall out of solution, providing an easy method for the recovery of ELP-tagged fusion proteins. Self-cleaving of the ELP tag and subsequent precipitation of the cleaved tag delivers the active purified product with no additional amino acids at its termini. The second method involves the co-expression of polyhydroxybutyrate (bioplastic) granules in the host cell. In this case, the granules act as an affinity carrier, while the co-expressed target protein is tagged with a self-cleaving granule-binding protein. Cell lysis and washing of the granules purifies the bound fusion protein, which is then released from the washed granules by self-cleaving of the granule-bound tag. Results are presented for the purification of over a dozen recombinant proteins expressed in E. coli. Shake flask yields of cleaved targets are typically on the order of tens of milligrams per liter to over 100 milligrams per liter depending on the target and method. The recovered proteins all have shown activity and purity comparable to commercially available preparations, and in some cases the ELP tag has been shown to increase solubility of difficult proteins. Thus this technique can be trivially applied to a wide variety of protein targets, and is likely to have a significant impact in protein purification for industrial and research purposes. More recent work has focused on the scale-up of these methods using crossflow filtration to separate aggregates from soluble contaminants. Further, an economic analysis indicates substantial savings in the cost of purification relative to conventional methods.