基于两性离子交换介质的蛋白质复性与分离纯化过程集成的研究

Protein refolding operation for active protein recovery is a crucial step in the production of recombinant proteins. The key strategy to enhance protein refolding is to inhibit protein aggregation in the refolding process. It has been reported that like-charged ion-exchange media in a refolding solution can greatly inhibit lysozyme aggregation and enhance lysozyme refolding yield. However, the mechanism of protein refolding facilitated by like-charged ion-exchange media is not clear, and the refolded protein can not be separated or purified by the like-charged ion-exchange media after refolding. In this project, the mechanical analysis of facilitated protein refolding and the integration of protein refolding, separation and purification will be achieved based on amphoteric ion-exchange media. The interaction between amphoteric ion-exchange media and protein molecules at refolding pHs will be studied by systematical methods including comprehensive thermodynamics, kinetics, and molecular dynamics simulations, and the theory of “protein refolding facilitated by amphoteric ion-exchange media” will be proposed. The separation and purification of inclusion bodies (IBs) after their refolding with amphoteric ion-exchange media will be studied at adsorption pHs, to reveal key factors for the integration of IB refolding, separation and purification based on amphoteric ion-exchange media, and to establish the mathematical model that explains the relation between IB properties and these key factors. This project will form the scientific and technological bases for integration of IB refolding, separation and purification.

蛋白质体外折叠复性是利用基因重组技术生产重组药物蛋白质的关键步骤，如何有效抑制蛋白质聚集，是高效复性的关键。有研究表明，与溶菌酶带有同种电荷的离子交换介质能够有效抑制溶菌酶聚集、提高复性收率。然而，该同电荷离子交换介质促进蛋白质复性机理尚不清晰，且其在促进蛋白质复性后不能分离纯化蛋白质。因此，本项目利用两性离子交换介质解析介质促进蛋白质复性机理，并在复性后对蛋白质进行分离纯化。运用实验研究与分子模拟相结合的方法，在复性pH下，从蛋白质复性收率、折叠动力学、热力学等方面，研究两性离子交换介质与蛋白质之间相互作用，建立两性离子交换介质“促进蛋白质复性”理论。在吸附pH下，研究两性离子交换介质对包含体蛋白质分离纯化作用，揭示影响包含体蛋白质复性与分离纯化过程集成的关键因素，并建立数学模型解析包含体蛋白质性质与上述关键因素之间关系，为指导包含体蛋白质复性与分离纯化过程集成提供理论基础和技术支持。

蛋白质体外折叠复性是利用基因重组技术生产重组药物蛋白质的关键步骤。蛋白质淀粉样聚集是引起帕金森症、阿尔茨海默症、Ⅱ型糖尿病等疾病的主要原因。有效抑制蛋白质聚集，既是高效复性的关键，也是治疗蛋白质构象病的合理路径。研究表明，某些固相材料能够抑制蛋白质聚集，且静电相互作用在其中发挥一定作用，然而其抑制机理尚不清晰，阻碍了高效聚集抑制剂的开发。针对此关键问题，本项目以胰岛素为模型蛋白质，以胰岛素聚集过程为研究对象，通过配基修饰制备了一系列带正电荷和负电荷的氧化石墨烯，将其应用于抑制胰岛素聚集的过程中，考察了带正电荷和负电荷的氧化石墨烯基于静电相互作用对胰岛素聚集过程的影响，总结了抑制剂的性质在胰岛素聚集过程中的作用规律，证实了带正电荷和负电荷的氧化石墨烯能够有效抑制胰岛素聚集，揭示了带正电荷和负电荷的氧化石墨烯基于静电相互作用的抑制胰岛素聚集的机理，提出了“相反电荷抑制聚集”理论和“相同电荷抑制聚集”理论，总结了高效蛋白质聚集抑制剂的设计思路。本项目的研究成果为开发新型高效蛋白质聚集抑制剂，治疗蛋白质构象病提供了理论和技术支持。