2D crystals of CotY as a scaffold for the development of novel nano-biomaterials

Highly organized macromolecular protein assemblies are attracting increasing interest as next-generation biomaterials due to their specific structural and functional diversity, offering potential applications in biocatalysis, drug delivery, and vaccine development. Even more possibilities open up if proteins are used as scaffolds to add entirely new functions by attaching enzymes, antigens or foreign proteins. In this work, we attempted to use CotY, a Bacillus subtilis spore coat protein, as a scaffold that, when produced in E. coli , forms 2D crystalline structures characterized by high stability. However, genetic fusion, a method for displaying foreign proteins on the surface of CotY crystals, adversely affected the formation of this macromolecular structure. To overcome this limitation, we employed the SpyTag003/SpyCatcher003 bioconjugation system to engineer CotY variants suitable for modular protein attachment. Several CotY-SpyTagged constructs were generated by inserting the SpyTag peptide at different positions, and their functionality was assessed using GFP and α-amylase fused to SpyCatcher as conjugation partners. Analysis of fluorescence intensity and enzymatic activity identified that the CotY156-SpyTag and C-terminal fusion of CotY and SpyTag were the most effective alternatives. These hybrid proteins also retained their ability to form lattices. Our findings demonstrate that self-assembling CotY-SpyTag proteins provide a versatile, user-friendly platform for immobilizing proteins of various sizes, enabling covalent attachment of multiple proteins and thus offering a new tool for the development of functional biomaterials.