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

用于生物制药研究和制造纯化平台的高度可控自切割标签

• 批准号: 1264322
• 负责人: David Wood
• 金额: $ 24万
• 依托单位: Ohio State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-15 至 2017-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1264322&HistoricalAwards=false
• 关键词: Highly; Controllable; Self; cleaving; Tags

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.

1264322 Wood，David W.这种NSF生物技术，生化和生物量工程计划奖将支持开发超可控制的自我裂开的Inteins。 intein是在医学研究和生物技术中使用许多应用的自我切换的蛋白质元素。 这项工作最重要的是使用输入来生成自我切换的亲和力标签，这可以使快速而简单的纯化平台方法用于任意重组靶蛋白。 这些输入的开发将采用有效的新遗传筛选采用理性蛋白质工程方法和进化方法。 特别是，理性工程方法将涉及将设计的变构金属结合位点插入内部结构，这将使内部切割反应由低水平的金属离子控制。 进化方法将涉及酵母表面显示，合理突变库设计和流动辅助细胞分类的组合，以鉴定具有优化温度和pH活性曲线的突变型连体。 然后，通过这些方法产生和优化的高度可控制的切割蛋白将在各种具有一系列常规亲和力纯化标签的蛋白质表达系统中证明。快速，可靠地纯化重组蛋白的能力对于生物药物开发和制造以及纯生物学和医学研究至关重要。 解决此问题的一种方法是使用自我切换的亲和力标签。 这些标签充当“分子钩”，以简化给定靶蛋白的纯化，然后一旦纯化完成，然后将其移开。 然而，出于多种技术原因，现有的自我切换标签方法在很大程度上仅限于细菌系统中表达的简单蛋白质。 这项工作将开发下一代自我裂开的内素，可以应用于任何表达宿主的非常简单且高度可靠的蛋白质纯化方法的开发。 尤其是，提出的Inteins将允许在哺乳动物和其他真核表达系统中应用自我裂解的亲和力标签方法，在过去，现有的Inteins过早切割过去使这是不切实际的。 由于这些宿主对于复杂的人糖蛋白的生产至关重要，因此预计这些内膜有望加速生物药物研究，并最终可能会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极好的培训机会，并将用作刺激高中生和本科生在这些领域中兴趣和专业知识的平台。