Programmable protein topology via SpyCatcher-SpyTag chemistry in one-pot cell-free expression system

Protein-based biomaterials play a significant role in biomedicine and biocatalysis due to their intrinsic biocompatibility and biodegradability. Topological biomaterials show certain advantages without changing the wild-type sequence of the protein, such as unique biofunctions and exceptional stabilities. However, the tuning for the synthesis and assembly of topological protein materials was limited. In this study, we combined the SpyCatcher/SpyTag (SC/ST) chemistry and proposed a cell-free one-pot transcription-translation-assembly system for flexibly regulating the production of topological protein materials. Dimers, trimers, and multimers of proteins with topological structures were designed. Next, the cell-free system was optimized by adjusting the magnesium ion concentration and the molar ratio of different plasmids to obtain the greatest degree of polymerization. The optimal Mg ion concentration was finally determined to be 15 mM, and their most appropriate plasmid molar ratios (SC:ST) were 7:3 for dimers, trimers, and multimers. Finally, based on the topological structure of the polymer, the function was verified with the fusion of xylanase, and it was found that the xylanase activity of the polymer was three times that of the xylanase monomer. Universally, the cell-free system in this study can be used to synthesize protein materials with different topologies based on various covalent or non-covalent methods, and it is likely to have potential in topological structure exploration and bioapplications.