Highly Controllable Self-cleaving Tags for Biopharmaceutical Research and Manufacturing Purification Platforms

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1264322 Wood, David W. This NSF Biotechnology, Biochemical and Biomass Engineering program award will support the development of hyper-controllable self-cleaving inteins. Inteins are self-cleaving protein elements with many applications in medical research and biotechnology. Most central to this work is the use of inteins to generate self-cleaving affinity tags, which enable rapid and simple purification platform methods for arbitrary recombinant target proteins. The development of these inteins will employ rational protein engineering approaches and evolutionary methods with a powerful new genetic screen. In particular, the rational engineering approach will involve the insertion of a designed allosteric metal binding site into the intein structure, which will allow the intein cleaving reaction to be controlled by low levels of metal ion. The evolutionary methods will involve a combination of yeast surface display, rational mutant library design, and flow assisted cell sorting to identify mutant inteins with optimized temperature and pH activity profiles. The highly controllable cleaving inteins generated and optimized via these methods will then be demonstrated in a variety of protein expression systems with a range of conventional affinity purification tags.The ability to rapidly and reliably purify recombinant proteins is critical for biopharmaceutical development and manufacturing, as well as for pure biological and medical research. One way to approach this problem is through the use of self-cleaving affinity tags. These tags act as 'molecular hooks' to simplify the purification of a given target protein, and then remove themselves once the purification is complete. For a variety of technical reasons, however, existing self-cleaving tag methods have been largely limited to simple proteins expressed in bacterial systems. This work will develop a next-generation self-cleaving intein, which can be applied to the development of very simple and highly reliable protein purification methods for any expression host. In particular, the proposed inteins will allow self-cleaving affinity tag methods to be applied in mammalian and other eukaryotic expression systems, where premature cleaving by existing inteins has made this impractical in the past. Because these hosts are critical for the production of complex human glycoproteins, these inteins are expected to accelerate biopharmaceutical research, and may ultimately decrease the manufacturing costs of these critical drugs. In addition, this work will provide an excellent training opportunity for students interested in biotechnology, biopharmaceuticals and medicine, and will be used as a platform for stimulating interest and expertise in these areas among high school students and undergraduates.