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

作　　者：闫治华 刘娜 刘远声 许成钢 YAN Zhi-Hua;LIU Na;LIU Yuan-Sheng;XU Cheng-Gang(College of Veterinary Medicine,College of Animal Science and Technology,Zhejiang Agriculture and Forestry University,Hangzhou 311300,China;Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education,Institute of Biotechnology,Shanxi University,Taiyuan 030006,China)

机构地区：[1]浙江农林大学动物科技学院动物医学院兽医系,杭州311300 [2]化学生物学与分子工程教育部重点实验室,山西大学生物技术研究所,太原030006

出　　处：《中国生物化学与分子生物学报》2022年第12期1694-1702,共9页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.32170053);山西省自然科学基金(No.201901D211195);浙江农林大学科研发展基金(No.2022LFR065)资助。

摘　　要：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.

关 键 词：解纤维梭菌 纤维小体 骨架蛋白 肽-蛋白质共价偶联技术 分子设计与固定化组装 

分 类 号：Q71[生物学—分子生物学]