The ability to directly and uniformly immobilize proteins onto surfaces in robust fashion with full retention of activity is a highly coveted goal in the fields of biodetection and bioengineering. More efficient attachment methods suitable for a wide range of polypeptides will lead to the development of high-value applications, devices and systems that will significantly impact research, drug discovery, diagnostics, biodetection, and biomimetics. The proposed goal of controlling molecular orientation during protein immobilization on inorganic surfaces is essential to emerging applications of biomimetics including biomineralization and nano-architecture. In this project, we achieve our objective through the development of fusion proteins with gold-binding peptides that confer high-affinity gold binding under mild conditions. In phase I we chose Protein A and Streptavidin as fusion partners because they have established commercial value in numerous applications. Compared to conventional procedures to immobilize polypeptides to gold, e.g., adsorption and chemical methods, our affinity tag for gold approach provides greater stability, reduced interference, and high specific activity of fusion partners, all of which contribute to higher sensitivity of biodetection. We achieved proof of concept in phase I and propose to complete the characterization and optimization of gold-binding of our fusion proteins with the goal of developing a high-affinity tag for immobilizing virtually any polypeptide on gold. In addition, we propose to develop prototype surface plasmon resonance biosensors using our fusion proteins for several commercial applications. Also, we propose to produce specific mutant forms of the gold-binding peptide to better understand the structure/activity relationship of gold binding with the goal of designing more efficient affinity tags for specialized applications using unique gold substrates, e.g., nanogold particles. Successful completion of phase II studies will produce high-value reagents and prototype sensor applications with strong commercial potential and significant societal benefits. In the long term, achieving our goals will significantly impact the areas of surface chemistry, biodetection, and biomaterials. ? ?
