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

1264322 Wood, David W.这项NSF生物技术、生化和生物质工程项目奖将支持超可控自裂蛋白的发展。蛋白是一种自切割的蛋白质元件，在医学研究和生物技术中有着广泛的应用。这项工作的最核心是使用内部蛋白产生自切割亲和标签，这使得对任意重组靶蛋白的快速和简单的纯化平台方法成为可能。这些蛋白的开发将采用合理的蛋白质工程方法和具有强大的新基因筛选的进化方法。特别是，合理的工程方法将涉及在内部结构中插入设计的变弹性金属结合位点，这将允许内部切割反应由低水平的金属离子控制。进化方法将包括酵母表面展示、合理的突变文库设计和流动辅助细胞分选，以确定具有优化温度和pH活性谱的突变蛋白。通过这些方法生成和优化的高度可控的切割蛋白将在一系列常规亲和纯化标签的多种蛋白质表达系统中得到验证。快速可靠地纯化重组蛋白的能力对于生物制药开发和制造以及纯生物和医学研究至关重要。解决这个问题的一种方法是使用自切割亲和标签。这些标签就像“分子钩子”一样，可以简化特定目标蛋白的纯化，然后在纯化完成后将其移除。然而，由于各种技术原因，现有的自切割标签方法在很大程度上局限于细菌系统中表达的简单蛋白质。本工作将开发新一代自裂蛋白，可用于开发非常简单和高度可靠的蛋白纯化方法，适用于任何表达宿主。特别是，所提出的内含物将允许自切割亲和标签方法应用于哺乳动物和其他真核表达系统，在这些系统中，现有内含物的过早切割在过去已经使这种方法不切实际。由于这些宿主对复杂的人类糖蛋白的生产至关重要，这些蛋白有望加速生物制药研究，并最终降低这些关键药物的制造成本。此外，这项工作将为对生物技术、生物制药和医学感兴趣的学生提供一个很好的培训机会，并将作为一个平台，激发高中学生和本科生对这些领域的兴趣和专业知识。

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.