Protein design for multi-material antifouling coatings

Summary

Biofouling at solid-liquid interfaces is a major challenge for molecular biosensors, implantable biomaterials, and other surfaces exposed to biological fluids. A common solution is to coat surfaces with hydrophilic polymer brushes that resist fouling. Traditionally, these brushes are chemically synthesized and covalently attached. In this thesis, we explore an alternative approach: designing genetically encoded proteins that self-assemble into antifouling brushes. These proteins follow a modular B-M-E structure, where B is a surface-binding domain, M promotes multimerization, and E is a hydrophilic, disordered polypeptide that provides antifouling function. In Chapter 2, we test the modularity of B-M-E proteins by varying both the B and E domains. By replacing the original silica-binding B domain with a gold-binding peptide, we successfully retarget the proteins to gold surfaces. We also modify the E domain using zwitterionic elastin-like polypeptides (ELPs), producing a new family of functional antifouling proteins for gold. Chapter 3 focuses on polystyrene-binding B-M-E proteins. Experimental data and simulations show these brushes are less stable on polystyrene than on gold or silica, and they are gradually displaced by serum proteins, indicating a need for further optimization. In Chapter 4, we aim to identify optimal E block sequences by screening hydrophilic and disordered protein domains known for enhancing solubility or serum stability. By varying the length of the E domain, we find around 100 amino acids to be optimal. We then compare different sequences of this length and identify one—E = [(GAGAIP)₃-(GAGEIP)]₄—that performs comparably to a synthetic tetraethylene glycol brush on gold surfaces. Chapter 5 explores functionalization of B-M-E brushes using SpyCatcher/SpyTag chemistry. We show that these domains can mediate specific reactions both in solution and when assembled on surfaces, enabling targeted attachment of molecules like GFP. Some constructs show partial degradation of the SpyTag, but still retain functionality. Finally, Chapter 6 discusses the benefits and current limitations of protein-based antifouling coatings. While promising for modular, biocompatible surface modification, challenges remain in optimizing stability and expression. Future work may focus on expanding the library of E domains, improving performance on diverse materials, and enabling multifunctional coatings.