Technology Development for Recombinant Affinity Reagents

重组亲和试剂技术开发

• 批准号: 8335435
• 负责人: BRIAN KENNETH KAY
• 金额: $ 118.67万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-25 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8335435
• 关键词: Affinity; Ankyrin Repeat; Antibodies; Arizona; Biological; Biosensor; Cells; Chicago; Collaborations; DNA; Emulsions; Engineering; Evaluation; Feedback; Fibronectins; Goals; Human; Human Genome; Illinois; Immunoglobulin Variable Region; Laboratories; Molecular; Monoclonal Antibodies; Process; Property; Proteins; Proteome; Protocols documentation; Reagent; Recombinants; Research; Screening procedure; Set protein; Technology; Testing; Tissues; Universities; cost effective; design; improved; inhibitor/antagonist; repository; structural genomics; synthetic peptide; technology development; tool

We propose to form a research collaboration in which we improve upon the screening and evaluation technologies currently used to generate affinity reagents. We intend to refine the discovery process so that it is not only cheaper and faster than currently possible, but also so that we produce reagents that are more versatile and useful than current monoclonal antibodies. This Center will involve the laboratories of Drs. Brian Kay (University of Illinois at Chicago), Andreas Pluckthun (University of Zurich), and Michael Weiner (lllumina Corporation). We will focus on three types of recombinant affinity reagents: human single-chain Fragments of variable regions (scFv), Designed Ankyrin Repeat Proteins (DARPins), and fibronectin type III (FN3) monobodies. Dr. Aled Edwards (Structural Genomics Center) will supply us with 100 targets to which we will apply our technology improvements. We will also use synthetic peptides, corresponding to the N-termini and C-termini of a test set of proteins, as targets. Our research plan consists of three strategic goals. First, through molecular engineering we will generate versatile affinity reagents that can accomplish the many standard tasks of antibodies, as well as function as biosensors and inducible inhibitors of intracellular targets. Second, we plan to improve our current selection protocols by using emulsions to make them faster, smaller, cheaper, and better. Third, in addition to our own characterization, we will distribute affinity reagents to five beta-testers for their evaluation in biological applications and receive feedback on what additional properties should be engineered into the affinity reagents. Once we have prepared and vetted high quality affinity reagents, they will be made available through the DNA repository at Arizona State University and two reagent supply companies. The overarching goal of our research will be to optimize technologies that can be applied to the entire human proteome in a versatile, powerful, cost-effective, and high-throughput manner.

我们建议建立一种研究合作，在当前用于生成亲和力试剂的筛选和评估技术上，我们可以改进。我们打算完善发现过程，以便它不仅比当前更便宜，更快，而且还可以生产比当前单克隆抗体更通用和有用的试剂。该中心将涉及博士的实验室。布莱恩·凯（Brian Kay）（伊利诺伊大学的芝加哥大学），安德烈亚斯·普鲁克（Andreas Pluckthun）（苏黎世大学）和迈克尔·韦纳（Michael Weiner）（Lllumina Corporation）。我们将关注三种类型的重组亲和力试剂：可变区域的人类单链片段（SCFV）设计了Ankyrin重复蛋白（DARPINS）和Fibronectin型III（FN3）单体形成。 Aled Edwards博士（结构基因组学中心）将为我们提供100个目标，我们将采用技术改进。我们还将使用对应于测试蛋白的N末端和C末端的合成肽作为靶标。我们的研究计划包括三个战略目标。首先，通过分子工程，我们将生成多功能亲和力试剂，这些试剂可以完成许多抗体的标准任务，以及作为生物传感器和细胞内靶标诱导抑制剂的功能。其次，我们计划通过使用乳液使其更快，更小，更便宜且更好的方法来改善当前选择方案。第三，除了我们自己的特征外，我们还将向五个β-测试器分发亲和力试剂，以在生物应用中评估，并收到有关应在亲和力试剂中设计其他属性的反馈。一旦我们准备并审查了高质量亲和力试剂，它们将通过亚利桑那州立大学的DNA存储库和两家试剂供应公司提供。我们研究的总体目标是优化可以以多才多艺，功能强大，具有成本效益和高通量方式应用于整个人类蛋白质组的技术。