为探究生物统合加工嵌合纤维小体中重组蛋白粘连模块与对接模块的分子相互作用及其对嵌合纤维小体组装效率的影响,采用酿酒酵母细胞分泌表达支架蛋白和酶分子催化模块,在胞外通过非变性蛋白凝胶电泳和等温滴定量热法测定分析二级支架蛋白、纤维素酶分别与一级支架蛋白结合时的相互作用反应。结果显示,分别连接二级支架蛋白和纤维素酶蛋白的对接模块与粘连模块的亲和力常数增大,亲和力减小,组装反应为焓变驱动的放热反应并产生氢键,证明这些蛋白分子间亲和力减小是导致二级支架蛋白与一级支架蛋白组装效率较低的主要影响因素。
Abstract
In order to explore the molecular interaction between recombinant protein cohesin module and dockerin module and its effect on the assembly efficiency. In this study, scaffoldins and enzymes was secreted by Saccharomyces cerevisiae cells, and the interaction of secondary scaffolins and cellulases combined with primary scaffolin was determined and analyzed by non-denatured electrophoresis and isothermal titration calorimetry in vitro. The results showed that the affinity force between the dockerin module of secondary scaffold protein and cohesion module of cellulase protein decreased while the affinity constant (Kd) increased. The assembly reaction was driven by enthalpy change and hydrogen bonds are generated. The results proved that the decrease of intermolecular affinity of the proteins was the main factor that leads to the lower assembly efficiency of secondary scaffold proteins and primary scaffold proteins.
关键词
酿酒酵母 /
嵌合纤维小体 /
纤维素酶 /
自组装 /
蛋白相互识别 /
分子动力学
Key words
Saccharomyces cerevisiae /
designer cellulosome /
cellulase /
self-assembly /
protein mutual recognition /
molecular dynamic
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基金
国家自然科学基金（31971202）; 国家科技支撑计划（2019YFB1503802）
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