Affinity tag technology has become increasingly important for immobilizing proteins for high-throughput screening, for generating protein arrays, and for simplifying protein purification. There is a pressing need for economical and technically simple technologies to immobilize or purify proteins. None of the existing molecular tags used for affinity purification is ideal for both purification and immobilization of proteins. The avidin-biotin complex has the highest affinity interaction known in nature, and this characteristic has made the avidin-biotin technology the gold standard for immobilizing proteins. A recent advance in this technology has been the development of biotin-accepting peptides (AviTag) that can be fused to proteins and exploited for applications requiring protein immobilization such as high-throughput screening, generating protein arrays, staining and sorting cells, and analysis of protein-protein interactions using surface plasmon resonance. Unfortunately, current non-denaturing methods of purifying biotinylated proteins are relatively expensive and technically problematic. In the proposed study, we will use directed evolution, a powerful technique for identifying highly specific peptide ligands, to create a new, low cost, highly selective affinity purification modality to complement our existing technology for immobilization of biotinylated protein.
