Antibodies, most commonly IgGs, have been widely used as targeting ligands in research and therapeutic applications due to their wide array of targets, high specificity and proven efficacy. Many of these applications require antibodies to be conjugated onto surfaces (e.g. nanoparticles and microplates);however, most conventional bioconjugation techniques (e.g. EDC-NHS) exhibit low crosslinking efficiencies, hinder functionality due to non-site-specific labeling, and/or require protein engineering (e.g. cysteine handles), which can be technically challenging and time consuming. This can result in high costs, heteregeneous samples, and poor reproducibility. To overcome these limitations, we will recombinantly express Protein Z, which binds the Fc region of IgG, with an UV active non-natural amino acid benzoylphenyalanine (BPA) within its binding domain. Upon exposure to long wavelength UV light, the BPA will be activated and form a covalent link between the Protein Z and the bound Fc region of IgG. This technology will be combined with expressed protein ligation (EPL), which will allow for the introduction of a fluorophore and click compatible azide group onto the C-terminus of Protein Z during the recombinant protein purification step. This will enable crosslinked- Protein Z-IgG complexes to be efficiently and site-specifically attached to aza-dibenzycyclooctyne-modified microplates and nanoparticles, via copper-free click chemistry. This approach is cost-effective, easily scalable, and utilizes protein production techniques that are commercially viable. Site-specific labeling has not only been shown to improve the activity of various surface-bound antibody conjugates (e.g. immunoassays and nanoparticles) but also non-surface bound antibody conjugates (e.g. antibody-drug conjugates) and thus the approach proposed here is expected to have broad applicability.
The specific aims for the proposal are (1) Optimize the yield of photoreactive Protein Z expression and the efficiency of expressed protein ligation;(2) Optimize the photo-crosslinking efficiency between Protein Z and various IgG subtypes.
The goal of this proposal is to develop a simple, robust technique for the efficient and site-specific covalent attachment of IgG onto nanoparticles. The bioconjugate technique can also be utilized for the site-specific attachment of IgG onto microplates or to produce non-surface bound antibody conjugates (e.g. antibody-drug conjugates). Therefore, it is expected that this technology will have broad, far-reaching applicability.