Study on the properties of a dual-system-based protein scaffold for orthogonal self-assembly.

Protein scaffolds possessing the ability to efficiently organize enzymes to improve the catalytic performance, enzyme stability and provide an optimal micro-environment for biocatalysis. Here, SpyCatcher fused to the C-terminus of Treptavidin (a variant of streptavidin) to construct a chimeric tetramers protein scaffold (Tr-SC) with dual orthogonal conjugation moieties. The results showed that the expressed Tr-SC scaffold was an active tetramer with good stability under 80 °C and pH 6.5-8.5, which could bind 4 SpyTag-mCherry and 4 Biotin-EGFP. Tr-SC scaffold can bind 1-4 ligands alone under different conditions. The order in which protein scaffolds bind to proteins has little effect on the final complex structure. It is more difficult for SpyTag-mCherry than Biotin-EGFP to bind to Tr-SC, so incomplete conjugates of a hexameric complex composed of 2 SpyTag-mCherry and 4 Biotin-EGFP form when the molar ratio of scaffold and two ligands is 1:4:4. Therefore, it was suggest that the Tr-SC can first bind to excess SpyTag-protein and mixed with Biotin-protein to promote the formation of higher multimers. The results can be important reference for more extensive use of Tr-SC to construct heterologous protein polymers and assembly of heterologous enzyme molecular machine in vitro to carry on efficient cascade reaction in the future.