Principal Investigator K Wittrup
The main goal of this project is to develop single fibronectin Ig domains with binding affinity mimicking that of Protein A by a yeast-surface display combinatorial approach. The particular aim is to develop new purification methodologies, with emphasis on milder elution conditions to avoid antibody aggregation. The stability and binding properties of the Protein A mimics will be engineered by both directed evolution and rational design.
Affinity purification requires the availability of binding reagents with both the necessary molecular recognition properties (affinity, association rate, specificity, reversibility) and industrial processing properties (robustness to immobilization and multiple rounds of purification and regeneration). Protein A meets this need for monoclonal antibody purification, however falls short in requiring acidic elution conditions that partially denature and consequently cause the aggregation of many antibodies.
Alternative molecules often display enhanced robustness, resistance, stability and cost- efficient production as compared to their natural templates. The main goal of this sub- project is to develop single fibronectin Ig domains with binding affinity mimicking that of Protein A by a yeast-surface display combinatorial approach. The particular aim is to develop new purification methodologies with emphasis on milder elution conditions to avoid antibody aggregation. The stability and binding properties of the Protein A mimics will be engineered by both directed evolution and rational design. The Protein A mimics will be thoroughly characterized and used as alternative ligands for the affinity purification of antibodies from different species, classes and subclasses.
The thermal stability, stability to caustic treatment, and binding properties will be engineered by directed evolution, and then thoroughly experimentally characterized. These binders will also be used as secondary reagents for the development of standard immunoassays such as ELISAs, and immunofluorescence methods.