人工纤维小体骨架蛋白的分子设计与自组装

Molecular Design and Self-assembly of Cellulosomal Scaffoldin

纤维小体是厌氧微生物分泌的能够高效降解木质纤维素的一种多酶复合体。其骨架蛋白质的大量重组合成是人工纤维小体构建的关键。为了构建全长的结构复杂的能招募大量酶亚基的骨架蛋白质,本研究对解纤维梭菌Ruminiclostridum cellulolyticum骨架蛋白CipC进行分子模块化设计,并分别与肽-蛋白质共价偶联系统SpyTag/SpyCatcher和SnoopTag/SnoopCatcher融合表达。然后分别通过非固定化和固定化组装策略对人工纤维小体骨架蛋白质进行了初步组装。结果显示,SpyTag和SpyCatcher、SnoopTag和SnoopCatcher之间自发形成稳定的共价异肽键,而且SpyTag和SnoopCatcher,SnoopTag和SpyCacther之间无交叉反应。最终成功组装出含有不同数量黏附域的骨架蛋白质,实现骨架蛋白链的延伸。本研究为构建结构更复杂、活性更高的人工纤维小体提供了新的设计思路并奠定了其技术基础。

Anaerobic and celluloytic bacteria secret a high molecular weight multienzymatic complex called cellulosome to degrade lignocellulose. It is crucial that recombinant synthesis of cellulosomal scaffoldin for designing artificial cellulosome. Based on scaffoldin of R. cellulolyticum, we synthesize recombinant scaffoldin by modular design and self-assembly imploying by peptide/protein covalent coupling technology of SpyTag/SpyCacther and SnoopTag/SnoopCatcher. The results of covalent coupling experiment revealed that the stable covalent binding could be spontaneously formed between SpyTag and SpyCatcher, SnoopTag and SpyCacther and both interactions did not cross-react with each other, suggesting that the system of peptide/protein covalent coupling were successfully established in our lab. The artificial scaffoldins were then assembled by using two strategies. Scaffoldins with different number of cohesions were firstly assembled by using various purified modules, by nonimmobilized self-assembly. Furthermore, a pentameric protein was successfully assembled on column by immobilized self-assembly. This study provides a new design idea for the construction of artificial cellulosome that can efficiently degrade lignocellulose.