Adsorption of protein onto dye-ligand affinity adsorbent: Molecular dynamics simulation and experimental study

亲和色谱是一种基于高度特异性分子识别的用于鉴定、纯化和分离大分子的成熟方法。染料配体亲和色谱法已广泛用于蛋白质纯化。固定化纺织品三嗪染料，特别是汽巴蓝3GA（CB）已被用作亲和层析工具，用于蛋白质纯化很长一段时间。CB能够结合多种蛋白质。然而，它与大量看似无关的蛋白质的相互作用不可避免地损害了其蛋白质结合特异性，并赋予这些分子一个严重的缺点。了解染料配体与蛋白质的结合及其特异性机制，对于提高纺织染料在蛋白质分离中的应用具有重要意义。在本计画中，我们将以先前研究过的血清白蛋白与血红蛋白为模式蛋白，探讨其在染料配体亲和层析介质上的吸附。由于缺乏实验方法，可以检测吸附剂孔内的动态吸附过程，分子动力学（MD）模拟与粗粒和全原子模型将在这项研究中首次使用研究染料配体亲和色谱吸附蛋白质在分子尺度上。在分子动力学模拟结果和前期实验研究的基础上，根据分子动力学模拟的结合机理，合理设计条件，进行了这些蛋白质在染料配体亲和吸附剂上的单吸附和双吸附实验。分子动力学模拟和吸附实验的结合将从微观和宏观两个角度全面理解亲和吸附的结合和具体机制。这一认识为设计和筛选新的亲和染料配体，以及设计、优化和控制蛋白质的染料配体亲和层析奠定了坚实的基础。

Affinity chromatography is a well-established method for the identification, purification, and separation of macromolecules based on highly specific molecular recognition. Dye ligand affinity chromatography has been widely utilized for protein purification. Immobilized textile triazine dyes, particularly Cibacron blue 3GA (CB) has been used as affinity chromatography tools for protein purification for a long time. CB is able to bind various proteins. However, its interaction with a large number of seemingly unrelated proteins inevitably compromises its protein binding specificity and endows these molecules with a serious drawback. For an improved application of textile dyes in the separation of proteins, it is very important to understand the binding and specific mechanisms between dye ligand and proteins. In this project, serum albumin and hemoglobin which have been investigated in our previous works will be further studied as model proteins to investigate their adsorption onto dye ligand (CB) affinity chromatographic media. Due to the lack of experimental method that can detect the dynamic adsorption process within adsorbent pores, molecular dynamics (MD) simulations with both coarse grained and all atom models will be used for the first time in this study to investigate the dye ligand affinity chromatographic adsorption of proteins on a molecular scale. On the basis of the results of MD simulations and the former experimental research, both single and binary adsorption experiments of these proteins onto dye ligand affinity adsorbent will be performed under conditions which are rationally designed according to the binding mechanism learned from the MD simulations. A combination of MD simulations and adsorption experiments will give a comprehensive understanding of the binding and specific mechanisms of the affinity adsorption from both microscopic and macroscopic point of view. This understanding is a profound basis for the design and screening of new affinity dye ligands, as well as the design, optimization and control of the dye ligand affinity chromatography of proteins.