静电排斥调控蛋白质复性的分子机理和应用

On the basis of our findings that protein refolding could be greatly facilitated by like-charged ion-exchangers and polyelectrolytes, as well as the understanding of the contributions of electrostatic repulsion of charged surfaces that induce oriented protein alignments that result in protein aggregating suppression, this project is proposed to study the molecular mechanisms of protein refolding modulated by the electrostatically repulsive charged surfaces as well as in the vicinity of polyelectrolytes. First of all, protein refolding in the presence of various charged particles and polyelectrolytes will be investigated to reveal the influences of important charge parameters on protein refolding and aggregation. Then, electrostatic interaction models for the folding protein in the electrostatically repulsive surfaces of charged particles and polyelectrolyte solutions will be established to examine the effects of protein structural parameters on the dynamics of electrostatic interaction energy, surface distance and orientation of protein molecules. At the same time, multi-scale molecular dynamics simulations will be carried out to study the conformational transition and intermolecular interactions of folding proteins in the electrostatically repulsive surfaces. The comprehensive researches will lead to the understanding of the molecular mechanisms of protein refolding modulated by the electrostatically repulsive surfaces. Based on the theoretical outcomes, novel charged media will be designed and synthesized for facilitating protein refolding, leading to the optimized medium structures in pore size, pore-size distribution, charged group chemistry and charge density. Finally, refolding and purification of like-charged proteins on ion-exchange column chromatography will be established and optimized to realize inclusion body protein refolding and purification. It is expected that the outcomes of this project will greatly contribute to the theoretical development of protein refolding, and lead to the invention of novel protein refolding technology. Therefore, this project is of theoretical importance and has great potential in protein refolding applications.

基于荷电粒子和聚电解质促进同电荷蛋白质复性作用的发现和表面静电排斥作用对抑制蛋白质聚集作用的认识，提出了静电排斥表面调控蛋白质复性的分子机理及其应用的研究构想。首先系统研究荷电粒子和聚电解质结构对蛋白质复性的影响规律，建立蛋白质在荷电粒子表面和聚电解质溶液等静电排斥纳米空间中的静电相互作用模型，研究蛋白质分子结构参数对静电相互作用和分子表面方位动态行为的影响；同时，利用多尺度分子动力学模拟研究蛋白质在静电排斥表面的构象转换和自相互作用，阐释静电排斥表面调控蛋白质复性的分子机理；在此基础上，设计合成新型结构的荷电粒子，研究荷电表面结构对蛋白质复性的影响，优化荷电介质性能参数；最后研究建立离子交换色谱辅助同电荷蛋白质复性和纯化方法，优化同电荷柱色谱复性过程，实现同电荷介质促进包含体蛋白质复性和纯化。本研究将发展蛋白质复性理论，创新蛋白质复性方法，具有重要理论研究意义和应用前景。

基因重组蛋白质的高表达常导致其在宿主细胞（如大肠杆菌等）内发生错误折叠和聚集，形成被称为包涵体的固体颗粒。因此，基因重组蛋白质药物的折叠复性是20多年来生物技术领域关注的焦点之一，也是基因重组包涵体蛋白质生产过程的瓶颈问题。深入开展蛋白质复性的基础研究，进而开发新型高效的蛋白质复性技术和过程，可以大幅度降低蛋白质药物的生产成本，从而占据蛋白质药物生产的制高点。本项目负责人前期研究发现，与蛋白质携带同种电荷的离子交换介质（荷电微球）可以显著促进蛋白质复性。基于此发现，本项目提出了系统研究静电排斥表面调控蛋白质分子结构转换和分子相互作用，通过解决相关基础科学问题，开发高效蛋白质复性促进剂的研究构想。经过系统研究，取得重要研究成果：（1）解析了盐和海藻糖等小分子对蛋白质变性过程热力学和动力学的影响，建立了荷电表面蛋白质和二肽分子结构转换和分子间相互作用模型，利用分子动力学模拟，考察了蛋白质结构转换行为，证明荷电表面上同电荷蛋白质（肽）的分子取向性及其导致的分子间相互作用显著降低，阐释了荷电表面对同电荷蛋白质聚集的抑制作用；（2）构建了高电荷密度介质的制备方法，包括次序接枝-电荷修饰方法和聚电解质接枝-二次电荷修饰方法，获得了极高电荷密度（1740 μmol/g）的纳米粒子，使介质添加量降低到3.3 μL/mL复性溶液；（3）建立了同电荷纳米粒子促进蛋白质复性与金属螯合亲和纯化的集成过程，实现了包涵体蛋白质的集成化复性和纯化。高电荷密度纳米粒子可使0.4 mg/mL包涵体复性收率提高100%，复性后加入镍离子即可发生蛋白质的金属螯合亲和吸附，纯度达到96%。研究成果深化了蛋白质结构转换和分子相互作用理论，开发了新型高效的蛋白质复性介质和方法，对促进蛋白质药物开发具有重要实际应用前景。共发表SCI收录学术论文20篇，获授权国家发明专利1项，申请国家发明专利3项。