Production and Immobilization of Antibody Fragments for Improved Affinity Purification

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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.