盘基网柄菌无细胞体系高效合成异源糖蛋白的探索性研究

The efficient and low cost synthesis of heterologous glycoproteins with correct glycosylation pattern is one of the most popular and key research problems in modern biological and pharmaceutical engineering. The single-celled slime mold, Dictyostelium discoideum (Dd), is easy to culture and possesses the complex cellular machinery required for orchestrating post-translational modifications similar to the one observed in higher eukaryotes such as human. Thus, this project is aimed at developing an efficient Dictyostelium discoideum cell-free protein synthesis system to effectively produce exogenous proteins with post-translational modifications by studying some key problems, such as the preparation of Dictyostelium discoideum extract, construction of cell-free expression vectors and energy recycling mechanism. Based on that, single-chain human choriogonadotropin (β-hCG), an important medical glycoprotein, will be expressed as a target protein in Dictyostelium discoideum cell-free system, and the synthesis and glycosylation mechanisms of β-hCG will be studied. Finally, the addition of E.coli extract into Dictyostelium discoideum cell-free system will be studied, and the possibility of constructing a cell-free hybrid system combining the high synthesis efficiency of E.coli and the glycosylation modification of Dictyostelium discoideum will be explored, which may improve both the synthesis efficiency and glycosylation level of target glycoproteins. In a word, it's possible to develop not only a new theory but also a universal cell-free expression system to express exogenous glycoproteins, which is promising in large scale production of complicated glycoproteins with medical interests.

如何高效低成本合成具有正确糖基化的异源糖蛋白是现代生物制药工程中最活跃和最核心的研究难题之一。基于盘基网柄菌单细胞世代容易培养和具有人类细胞类似的蛋白质后加工（糖基化）特性，本课题将通过盘基网柄菌无细胞抽提物制备、无细胞表达载体构建、能量再生体系等研究，建立一个高效表达外源蛋白并具有蛋白质翻译后修饰能力的盘基网柄菌无细胞蛋白合成体系。在此基础上，以人促绒毛膜性腺激素β亚基（β-hCG） 为研究对象，研究盘基网柄菌无细胞体系中这一示例糖蛋白的合成及翻译后糖基化修饰的机制。最后探索通过添加大肠杆菌无细胞抽提物等新手段，构建一个兼具大肠杆菌高蛋白合成能力和盘基网柄菌类人细胞糖基化能力的杂合体系，提高目标糖蛋白的合成能力和糖基化质量。总之，本项研究不仅能够建立体外高效合成异源糖蛋白理论基础，构建一个高效合成异源糖蛋白的无细胞体外通用型系统，而且对复杂药用糖蛋白大规模生产技术也具有重要推动作用

本项目构建了适合真核无细胞体系的表达载体，较系统考察了多种真核无细胞抽提物制备工艺和真核体外蛋白质表达条件，分别构建了盘基网柄菌家蚕丝腺和酿酒酵母无细胞蛋白质表达系统。其中家蚕丝腺和酿酒酵母体外表达系统具有较强的合成蛋白质能力，而盘基网柄菌体外合成系统相对表达蛋白质水平较差。利用以上自主构建的真核表达系统，实现多种蛋白质（hCG、荧光素酶、绿色荧光蛋白）的有效表达。自主开发了适用于24孔板的CECF无细胞蛋白表达装置，建立了同时交换同时反应的体外蛋白质合成新策略。与间歇操作相比，这一操作方式极大地提高了GFP和嗅觉受体蛋白odr的表达水平。最后利用自主构建的无细胞反应体系和部分体内表达体系的对比研究，开展了蛋白质翻译后的多种结构后修饰的研究，实现了胞壁酸糖肽结构单元的体外合成，胶原蛋白质中脯氨酸残基的羟基化以及体外水离子通道蛋白质中非天然氨基酸的渗入。本课题不仅建立多种真核体外蛋白质表达系统和新型操作模式，而且较深入探明体内外蛋白质合成过程中多种翻译后修饰的基本规律，这对进一步提高无细胞体系高效合成复杂蛋白质的研究具有重要的推动作用。