Nanoscopic Understanding of Protein Transport and Structure Dynamics in Charged Gels for Protein Chromatography

1032727 CartaIntellectual MeritThe广泛的目标，这项研究是一个基本的了解蛋白质运输带电荷的凝胶蛋白质色谱和新的矩阵的发展，将灵活的聚合物在一个刚性的支持矩阵。虽然蛋白质在中性凝胶中的扩散通常是很好理解的，但我们对吸附的生物分子如何在带相反电荷的基质中扩散的基本知识目前非常有限。在先前的工作中已经开发了通过将带电的葡聚糖聚合物接枝物并入多孔支持物中获得并表现出独特的蛋白质转运性质的新基质。然而，基本的传输机制是未知的，这阻碍了优化材料的开发和基本合理的模型对其的描述。因此，拟议的研究有三个主要目标：-通过动态光散射（DLS）确定葡聚糖接枝基质的结构和动态行为，以了解这些系统中聚合物链的波动和结构动力学。 使用单分子跟踪技术在纳米尺度上研究这些材料中的蛋白质传输，以确定单个蛋白质分子在带电聚合物功能化的基质中的迁移率。 开发多长度尺度的分子模型来描述聚合物功能化基质的结构和动态行为以及它们与蛋白质的相互作用，并使用这些结果来阐明目标2中的实验，并构建物理上精确的概念和现象学模型用于实际速率预测。这项研究的组成部分将提供一个尚未实现的水平的理解运输现象在这些复杂的媒体，基础上开发新的，更有效的材料，更广泛的影响：生物医学科学的进步提高了人们对用于预防、治疗和治愈严重疾病的新型生物药物的期望，事实上，新型蛋白质生物药物正在对人们的生活产生巨大影响。然而，分离和纯化这些生物分子由于其缓慢扩散而变得复杂，这限制了目前工业生物色谱的性能。拟议的项目有可能通过引入新的、更有效的固定相以及对传输现象前所未有的理解来改变生物制药的下游加工，从而增强对过程的理解和控制，并大大减少过程开发和设计所需的时间和精力。研究和教育将通过将该项目的结果纳入大学生的教育模块，通过加强研究生在实验和分子建模方面的协同活动的教育，以及通过每年在UVa举行的行业专业人士的蛋白质色谱短期课程来整合。外联将包括开发实验室演示，用于高中生向上的课程，通过促进少数民族本科生参与我们的短期课程，并从代表性不足的群体中招募研究生。

1032727CartaIntellectual MeritThe broad objectives of this research are a fundamental understanding of protein transport in charged gels for protein chromatography and the development of new matrices incorporating flexible polymers in a rigid support matrix. Although protein diffusion in neutral gels is generally well understood, our basic knowledge of how adsorbed biomolecules diffuse inoppositely charged matrices is currently extremely limited. New matrices obtained by incorporating charged dextran polymer grafts in porous supports and exhibiting unique protein transport properties have been developed in prior work. However, the underlying transport mechanisms are unknown, which prevents the development of optimized materials and their description with fundamentally sound models. Thus, the proposed research has three main goals:- To determine the structure and dynamic behavior of dextran-grafted matrices by dynamic light scattering (DLS) in order to understand fluctuations and structure dynamics of polymer chains in these systems.- To study protein transport in these materials at the nanoscopic scale using single molecule tracking techniques to determine the mobility of individual protein molecules in matrices functionalized with charged polymers.- To develop molecular models at multiple length scales to describe the structure and dynamic behavior of polymer functionalized matrices as well as their interactions with proteins, and use these results to elucidate the experiments in goal 2 and construct physically accurate conceptual and phenomenological models for practical rate predictions.Collectively, the components of this research will provide a yet unrealized level of understanding of transport phenomena in these complex media, the basis to develop new, more effective materials, and models to aid the design and optimization of chromatographic processes.Broader Impact:Advances in the biomedical sciences have raised expectations for new biologics-based drugs for the prevention, treatment, and cure of serious diseases and, indeed, new protein based biopharmaceuticals are having a huge impact on the lives of people. However, separating and purifying these biomolecules is complicated by their slow diffusion, which currently limits the performance of industrial biochromatography. The proposed project has the potential oftransforming downstream processing of biopharmaceuticals by introducing new, more effective stationary phases as well as an unprecedented level of understanding of transport phenomena, thereby enhancing process understanding and control as well as reducing dramatically the time and effort needed for process development and design. Research and education will be integrated by incorporating the results of this project in educational modules for college level students, by strengthening the education of graduate students with synergistic activities in experiments and molecular modeling, and through a Short Course in Protein Chromatography for industry professionals held annually at UVa. Outreach will include developing laboratory demonstrations for use in sessions for the Upward Bound program for high school students, by facilitating participation of minority undergraduate students in our Short Course, and by recruiting graduatestudents from underrepresented groups.