Production and Immobilization of Antibody Fragments for Improved Affinity Purification

本研究涉及新的策略来生产具有最佳结构的抗体多肽，以固定在不溶性载体上。每种方法的设计目的都是产生固定的单价抗体片段，其结合位点面向载体表面，并且通过多肽上远离结合位点本身的位点以高度特异性的方式与载体结合。这种方式的附着应该允许完全保留抗体结合活性，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个抗体应该会增加可以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，位点定向诱变将用于在多肽的所需位置合并单个附着位点。修饰后的多肽将在大肠杆菌中表达。在大肠杆菌中生产Fv片段可能被证明是当今杂交瘤技术的一种优越选择，杂交瘤技术经常是单克隆抗体生产和分离的限制因素。此外，定点诱变和随机诱变都可以快速获得具有变异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位点。

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.