(17f) Creating Proteins with Large Sizes and New Shapes for Biomedical Applications

Molecular structures greater than 100 kDa are extremely difficult to synthesize with high purity and yield, and the synthesis of large molecules often results in a heterogeneous product of undefined chemical composition. Here, we describe a synthetic strategy to produce protein-based materials that have masses greater than 500 kDa, exhibit non-linear connectivity, and are highly monodisperse. These biomaterials, which we term megamolecules, are synthesized by connecting fusion proteins with multifunctional crosslinkers that possess an irreversible inhibitor to form stable covalent adducts with a nucleophilic residue in the active site of the fusion protein. We used energy transfer to probe the structure of linear oligomers, up to a 300 kDa hexamer, and observed a non-globular structure with end-to-end distances greater than 10 nm. Further, we attached multiple antibody domains to linear and cyclic megamolecules, and observed increased antigen binding due to an avidity effect. We expect the ability to generate precisely defined structures will enable preparation of antibody conjugates, multispecific antibodies, and multivalent signaling molecules.