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

1264322伍德，大卫W。 这个NSF生物技术，生物化学和生物质工程计划奖将支持超可控自我切割内含肽的开发。 内含肽是一种具有自我切割功能的蛋白质元件，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含肽来产生自切割亲和标签，这使得能够快速和简单地纯化任意重组靶蛋白的平台方法。 这些内含肽的开发将采用合理的蛋白质工程方法和进化方法，具有强大的新的遗传筛选。 特别是，合理的工程方法将涉及将设计的变构金属结合位点插入内含肽结构中，这将允许内含肽裂解反应由低水平的金属离子控制。 进化方法将涉及酵母表面展示，合理的突变体库设计，和流动辅助细胞分选的组合，以确定具有优化的温度和pH活性曲线的突变内含肽。 通过这些方法产生和优化的高度可控的切割内含肽将在各种蛋白质表达系统中与一系列常规亲和纯化标签一起展示。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通过使用自切割亲和标签。 这些标签充当“分子钩”以简化给定靶蛋白的纯化，然后在纯化完成后将其自身去除。 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限于在细菌系统中表达的简单蛋白质。 这项工作将开发下一代自切割内含肽，它可以应用于开发非常简单和高度可靠的蛋白质纯化方法，适用于任何表达宿主。 特别地，所提出的内含肽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，其中现有内含肽的过早切割在过去已经使得这不切实际。 由于这些宿主对于复杂的人类糖蛋白的生产至关重要，这些内含肽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极好的培训机会，并将作为一个平台，激发高中生和本科生对这些领域的兴趣和专门知识。

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.