Biofunctional coatings via targeted covalent cross-linking of associating triblock proteins

A method for creating tailorable bioactive surface coatings by targeted cross-linking of network-forming CRC protein polymers is presented. The proteins are triblock constructs composed of two self-associating leucine zipper end domains (C) separated by a soluble, disordered central block (R) containing a cell or molecular binding sequence. The end domains preferentially form trimeric bundles, leading to the formation of a regular, reversible hydrogel network in a wide range of solution conditions. These hydrogel-forming proteins are useful for creating bioactive surface coatings because they self-assemble into networks, physically adsorb to a variety of substrate materials, and can be tailored to display numerous extracellular matrix (ECM)-derived peptides that interact with cells and biological macromolecules. Moreover, due to the close proximity of complementary Glu and Lys residues in the trimeric C bundles, these protein coatings can be stabilized in a targeted manner by covalent cross-linking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Here, we demonstrate that such EDC-cross-linked protein coatings are stable in cell culture media and maintain a significant level of biofunctionality when various ECM-derived peptides are embedded in the central soluble block of the proteins. First, we show that EDC cross-linking enables bioinert CRC protein coatings (those without embedded cell binding domains) to resist the adhesion of human foreskin fibroblasts in normal serum medium, but does not impair the ability of cross-linked coatings of CRC-RGDS (proteins with an embedded RGDS integrin binding domain) to promote cellular attachment, focal adhesion formation, and proliferation of these cells. Next, we show that the ability of cross-linked coatings of several new CRC-based proteins containing embedded heparin-binding sequences to bind biotinylated heparin is not significantly impacted over a range of EDC concentrations. The ability to target specific functional groups for covalent cross-linking is made possible by the specificity of protein-protein interactions and represents an important advantage of protein-based materials.