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

Abstract

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.