Production and Immobilization of Antibody Fragments for Improved Affinity Purification

抗体片段的生产和固定以改进亲和纯化

• 批准号: 9010955
• 负责人: Douglas Clark
• 金额: $ 13.01万
• 依托单位: University of California-Berkeley
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-08-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9010955&HistoricalAwards=false
• 关键词: Production; Immobilization; Antibody; Fragments; Improved

This research involves novel strategies to produce antibody polypeptides with optimal structures for immobilization to insoluble carriers. Each approach is designed to result in immobilized, monovalent antibody fragments with their combining sites facing out from the carrier surface, and attachment to the carrier will be achieved in a highly specific manner through a site on the polypeptide far removed from the combining site itself. Attachment in this fashion should allow complete retention of antibody binding activity and thus lead to improved techniques of affinity purification. In addition, immobilizing antibody fragments rather than the much large whole antibody should increase the number of antibody combining sites that can be immobilized on the carrier surface. In the case of Fv fragments, site-directed mutagenesis will be used to incorporate a single attachment site at a desired location on the polypeptide. The modified polypeptide will then be expressed in E. coli. Production of Fv fragments in E. coli may prove to be a superior alternative to present-day hybridoma technology, which is frequently the limiting factor in monoclonal antibody production and isolation. Moreover, both site- directed mutagenesis and random mutagenesis can be used to rapidly obtain antibody polypeptides with variant binding activities. Protein engineering may also be used to introduce pH or metal-regulated binding affinity into the combining site.

这项研究涉及到制备具有最佳结构的抗体多肽的新策略，以固定到不溶性载体上。每种方法都被设计成固定化的单价抗体片段，其结合部位面向载体表面，并且将通过远离结合部位本身的多肽上的部位以高度特异的方式附着到载体上。这种方式的附着应该允许抗体结合活性的完全保留，从而导致亲和纯化技术的改进。此外，固定抗体片段而不是大得多的整体抗体应该增加可固定在载体表面的抗体结合位点的数量。在Fv片段的情况下，定点突变将被用来在多肽上的期望位置结合一个单一的结合位点。修饰后的多肽将在大肠杆菌中表达。在大肠杆菌中生产Fv片段可能被证明是比目前的杂交瘤技术更好的替代技术，后者通常是生产和分离单抗的限制因素。此外，定点诱变和随机诱变均可用于快速获得具有不同结合活性的抗体多肽。蛋白质工程也可用于将pH或金属调节的结合亲和力引入结合部位。