Inteins and affinity resin substitutes for protein purification and scale up

Microbial Cell Factories Inteins and affinity resin substitutes for protein purification and scale up Mahmoud Reza Banki and David W Wood* 0 Address: Department of Chemical Engineering, A213 E-QUAD, Princeton University , Princeton, NJ 08544 , USA 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. - Introduction An important development in the area of recombinant protein purification has been the incorporation of selfcleaving protein elements into a variety of fusion-based purification systems [1-3]. These elements are derived from naturally occurring self-splicing inteins through various protein engineering strategies, and have been combined with conventional affinity tags in a variety of configurations to yield highly effective separations methods. Very recently, these elements have also been combined with non-conventional purification tags to yield "self-purifying" proteins, which can deliver highly purified native products using simple mechanical means without chromatographic methods [4,5]. This review will compare conventional affinity-tag methods, with and without proteolytic tag removal, to three newer methods based on self-cleaving purification tags. The three newer methods include a conventional affinity tag separation with a self-cleaving chitin-binding tag (the IMPACT system), a more recent method where the expression host produces a granular affinity matrix during fermentation (the PHB system), and a third in which the target protein is tagged with a reversibly-precipitating selfcleaving polypeptide (the ELP system). In particular, the advantages and disadvantages of each method will be compared, and the large-scale economics of each of these systems will be examined from a simple raw-materials cost standpoint. This simple analysis is intended to describe the relative merits of these methods, and to provide an initial benchmark for evaluating their potential future role in the large-scale manufacture of recombinant products. Conventional Affinity-Tag Methods Affinity fusion-based protein purification is a simple and now widely used method which takes advantage of the selective binding property of a genetically fused binding protein (tag) to purify a given target protein [6,7]. In place of physicochemical properties of the target protein, this technique relies on the specific binding of the affinity tag to an immobilized ligand. By exploiting this highly specific interaction, a single purification step can effectively isolate and purify a given target protein with ease. The development of numerous tags has further demonstrated the flexibility and potential of this method. Despite these strengths, however, the use of conventional gene-fusion affinity tags suffers from two main drawbacks. The first limitation arises from the requirement that the tag be removed in order to recover a native target protein. This is generally accomplished by enzymatically removing the tag from the purified target by the addition of an appropriate protease. To facilitate this procedure, the target sequence of the selected protease is genetically included between the tag and the target protein when the fusion is constructed, allowing specific cleaving to take place. Although this procedure is generally effective at laboratory scales, the cost of protease enzymes is prohibitive at manufacturing scale. In addition, yield losses can arise from incomplete cleaving or unexpected cleaving within the target, and the affinity tag and protease must also be separated from the cleaved target protein in a separate purification step. Both of these aspects increase the cost and complexity of the purification, while decreasing the yield. A second limitation arises from the equipment and consumable resin costs associated with these procedures. Conventional affinity resins typically consist of various cross-linked polymers, derivitized with appropriate ligands at the end of optimized spacer arms. Manufacturing costs for these resins are typically much higher than for ion-exchange and other chromatography resins, which can offset the appeal of the simpler affinity-based separation. A notable exception has been the widespread use of Protein A affinity columns in the purification of antibody therapeutics. However, this separation is limited to native antibodies, without the addition of a fusion tag. This suggests that conventional affinity tag methods may be attractive if tag removal can be simplified [8]. For these reasons, new methods which eliminate the need for protease treatment and expensive affinity resins are likely to make a significant impact on large-scale protein purification processes or high-throughput screening of protein libraries. The next two sections address the two drawbacks mentioned above and offer recently developed solutions. Self-cleaving Affinity Tags Inteins (INTervening protEINS) are naturally occurring protein sequences capable of post-translational self-excision from a host-intein precursor protein through a process known as "protein splicing" [9,10]. Several intein examples have been identified where the intein is capable of functioning outside of its native context, allowing these inteins to be developed for a variety of biotechnological applications. One of the most significant of these is the creation of self-cleaving protein elements that can be combined with conventional affinity tags to generate effective self-cleaving affinity tags [1-3]. A critical feature of these tags is their ability to release a target protein, fused either C or N-terminally to the tag, in response to a simple chemical or physical stimulus. The highly specific cleaving reaction thus allows the affinity tag to be removed without the addition of expensive protease, while at the same time preventing unwanted cleaving. An additional important advantage is that the cleaving reaction can be induced while the tagged target is bound to the affinity column, thus eliminating the need for subsequent removal of the cleaved tag. The first commercially available intein purification system was developed by New England Biolabs (NEB), and is based on a modified Saccharomyces cerevisiae vacuolar ATPase subunit A intein (Sce VMA intein) [1]. This intein possesses a particular mutation, leading it to exhibit N-terminal cleaving in the presence of 30 mM 1,4-dithiothreitol (DTT) or -mercaptoethanol over a wide pH range (5.5 9.0). This intein was combine (...truncated)