We introduce a new method for the purification of recombinant proteins expressed in Escherichia coli using self-cleaving elastin-like polypeptide (ELP) fusion tags without the need for affinity chromatography or proteolytic tag removal. Using this method we obtained high purity, activity and reasonable yields for ten diverse target proteins.

This work combines two well-established technologies to generate a breakthrough in protein production and purification. The first is the production of polyhydroxybutyrate (PHB) granules in engineered strains of Escherichia coli. The second is a recently developed group of self-cleaving affinity tags based on protein splicing elements known as inteins. By combining these technologies with a PHB-specific binding protein, a self-contained protein expression and purification system has been developed. In this system, the PHB-binding protein effectively acts as an affinity tag for desired product proteins. The tagged product proteins are expressed in E. coli strains that also produce intracellular PHB granules, where they bind to the granules via the PHB-binding tag. The granules and attached proteins can then be easily recovered following cell lysis by simple mechanical means. Once purified, the product protein is self-cleaved from the granules and released into solution in a substantially purified form. This system has been successfully used at laboratory scale to purify several active test proteins at reasonable yield. By allowing the bacterial cells to effectively produce both the affinity resin and tagged target protein, the cost associated with the purification of recombinant proteins could be greatly reduced. It is expected that this combination of improved economics and simplicity will constitute a significant breakthrough in both large-scale production of purified proteins and enzymes and high-throughput proteomics studies of peptide libraries.Keywords: protein purification; protein expression; polyhydroxybutyrate; intein; self-cleaving affinity tag Advances in protein expression systems have made possible the production of virtually any peptide product in at least one of a variety of host cells. Once expressed, however, these products must be purified to allow their study or use. Thus, the rapid and economical purification of recombinant proteins represents a persistent challenge in the field of biotechnology. Protein purification typically involves several chromatographic steps, each of which must be individually optimized for each product protein. Each step can be costly and timeconsuming, and inevitably decreases the final yield of the product (Freitag and Horvath 1996). This complexity can delay research on new proteins at the laboratory scale, and is becoming more significant with the completion of several genome sequencing projects. In the large-scale manufacture of recombinant proteins for industrial and therapeutic use, downstream purification is very costly and can account for up to 80% of the total production cost (Hearn and Acosta 2001). Although these costs may be acceptable for high-value therapeutics, the development of generic biologics will demand more cost-effective manufacturing strategies. The development of simple and reliable methods for Reprint requests to: David Wood, Department of Chemical Engineering, Princeton University, A417 Engineering Quadrangle, Olden Street, Princeton...

The development of self-cleaving fusion-tag technology has greatly simplified the purification of recombinant proteins at laboratory scale. The self-cleaving capability of these tags has recently been combined with additional purification tags to generate novel and convenient protein purification methods at a variety of scales. In this review, we describe some of these methods, and provide a rudimentary economic analysis of hypothetical large-scale applications. This work is expected to provide a rough outline for the evaluation of these methods for large-scale bioprocessing of a variety of products.

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