Cloning, Expression, and Purification of Ovalbumin and Its Variants

Abstract

Biotherapeutics, as exemplified by several monoclonal antibodies and therapeutic cytokines are increasingly getting popular for the treatment of a range of inflammatory diseases and cancers. A major challenge for the pharmaceutical industry has been to maintain them in biologically active form for extended periods. One of the major reasons for the loss of biological activity of a therapeutic protein is its aggregation that may occur during its production, transport, and storage. The aggregation of a therapeutic protein compromises its efficacy in several ways, one by reducing the availability of active monomers and secondly by generating neutralizing antibody response. An immunogenic response, commonly reported in the case of monoclonal antibody aggregates has been primarily ascribed to their effective uptake, processing, and presentation by dendritic cells. The uptake of a protein antigen by dendritic cells in an aggregated form drives their robust activation and maturation when compared to a monomeric antigen. Further, some reports suggest that the size and shape of the protein aggregates have a bearing on the dendritic cell and consequently B and T cell response. Protein aggregation, a challenge for monoclonal antibodies (and therapeutic cytokines) could be seen as an opportunity to enhance the immunogenicity of certain vaccine candidate antigens. The process of protein aggregation (or oligomerization) may be controlled by employing standard biophysical methods for the discovery of novel biophysical entities. The protein antigens in a supramolecular form are expected to stay at the site of injection for extended periods thereby circumventing the need for an adjuvant (alum). Further, an immunogen when administered in a supramolecular form would release oligomers of varying size and shape. Therefore, it would be interesting to draw a correlation between the biophysical form an antigen acquires and the immune response that it generates. In the proposed study, I will be harnessing the potential of multimerizing motifs to induce the multimerization of ovalbumin, a model antigen. Further, I will characterize the immune response to multimerized ovalbumin as a proof of concept that multimerization of protein antigens can remarkably enhance their uptake and presentation by antigen-presenting cells. Consequently, immunogenicity and efficacy of currently available sub-unit vaccines and to develop of newer sub-unit vaccines