Incorporation of azide groups into an engineered Fn3 scaffold protein for site-specific, bioorthogonal conjugation

Protein bioconjugates have a wide range of applications, including for targeted drug delivery and in vivo diagnostics. Expanding the chemistry available to link protein to cargo remains a critical consideration for the development of novel bioconjugates for unmet needs. Currently, most FDA-approved protein bioconjugates are based on the antibody structure, which constrains the available conjugation chemistries and applications. Alternative scaffold proteins have potential as targeting molecules that advance the synthetic possibilities. Here, we report two methods of high-yield incorporation of azide-containing groups into an Fn3 domain protein scaffold to enable click chemistry conjugation. One approach incorporates noncanonical amino acid azidohomoalanine during recombinant protein expression, and the other method develops a linker system using a unique thiol residue in the Fn3 structure. These synthetic approaches enabled orthogonal, dual labeling. The resulting conjugates retain functionality in receptor binding assays, validating these methods for diverse applications in engineering scaffold proteins for bioconjugate applications.