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.
摘要：
蛋白质支架具有有效组织酶以提高催化性能的能力，酶的稳定性，为生物催化提供最佳的微环境。这里,SpyCatcher与Treptavidin（链霉亲和素的变体）的C端融合，以构建具有双正交缀合部分的嵌合四聚体蛋白支架（Tr-SC）。结果表明，表达的Tr-SC支架是活性四聚体，在80°C和pH6.5-8.5下具有良好的稳定性，可以结合4个SpyTag-mCherry和4个生物素-EGFP。Tr-SC支架可以在不同条件下单独结合1-4个配体。蛋白质支架与蛋白质结合的顺序对最终的复杂结构几乎没有影响。SpyTag-mCherry比生物素-EGFP更难与Tr-SC结合，因此,当支架和两个配体的摩尔比为1:4:4时,由2个SpyTag-mCherry和4个生物素-EGFP组成的六聚体复合物的不完全缀合物形成。因此,建议Tr-SC可以首先与过量的SpyTag蛋白结合并与生物素蛋白混合以促进更高多聚体的形成。研究结果可为今后更广泛地利用Tr-SC构建异源蛋白聚合物和体外组装异源酶分子机进行高效级联反应提供重要参考。
