(789b) Engineering Proteins for Bioorthogonal Surface Conjugation of Gold Nanoparticles

Julia G. Fraseur^a, Katherine N. Clayton ^b, Tamara L. Kinzer-Ursem^a

^aWeldon School of Biomedical Engineering, Purdue University^bSchool of Mechanical Engineering, Purdue University

As the field of bionanotechnology is rapidly expanding, researchers are taking advantage of nanoparticle properties for the design of vehicles for nanomedicine, therapeutics, and biosensing. Key to the advancement of the field are the development of techniques to bind biomolecules of interest to nanoparticles to produce stable, highly bio-active nanoparticle conjugates. We have developed a novel method for in situconjugation of N-terminal azide-tagged proteins onto gold nanoparticle surfaces with consistent orientation, low aggregation, and high levels of activity.

In this work we selectively label proteins of interest co-translationally in recombinant expression systems using a chemoenzymatic tagging method. This method covalently labels protein with azide functionality that provides a handle for a rapid, bioorthogonal conjugation to nanoparticles via the azide-alkyne cycloaddition reaction, a popular â??clickâ? chemistry. Simultaneously, we have developed a method for functionalizing streptavidin-coated gold nanoparticles with a heterobifunctional PEG terminated with biotin and dibenzocyclooctyne (biotin-PEG4-DBCO) in order to conjugate the azide-tagged protein covalently to the nanoparticle surface. This chemistry allows for proteins to be consistently oriented onto the gold nanoparticle regardless of mixture complexity. With the azide tag, our target protein can be conjugated to the particle from a purified state or cell lysate. Although aggregation of nanoparticles post-conjugation can be a common occurrence when working with nanoparticles, we have optimized our methods such that protein-conjugated nanoparticles remain monodisperse post-conjugation. Furthermore, we show high levels of protein activity on the nanoparticles, ensuring that neither our tagging method, nor our conjugation methods inhibit protein functionality.

Here we will discuss the underlying methodology of protein tagging and nanoparticle conjugation by click chemistry. This technique streamlines the conjugation process and provides an opportunity for the successful functionalization of target proteins to gold nanoparticles directly from cell lysate. Future applications of these methods include characterization of protein-protein interactions on nanoparticles, biomarker detection, biosensing, and molecular diagnostics.