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

蛋白质色谱带电凝胶中蛋白质运输和结构动力学的纳米级理解

• 批准号: 1032727
• 负责人: Giorgio Carta
• 金额: $ 30.3万
• 依托单位: University of Virginia Main Campus
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-15 至 2014-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1032727&HistoricalAwards=false
• 关键词: Nanoscopic; Understanding; Protein; Transport; Structure

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.

1032727细节值得这项研究的广泛目标是对蛋白质色谱的带电凝胶中蛋白质转运的基本理解，以及在刚性支持矩阵中掺入柔性聚合物的新矩阵的开发。尽管通常对中性凝胶中的蛋白质扩散进行了充分的了解，但我们对吸附的生物分子如何不受控制的矩阵的基本知识目前非常有限。通过将带电的葡萄糖聚合物移植物掺入多孔载体和表现出独特的蛋白质转运特性的新矩阵已在先前的工作中开发出来。但是，基本的运输机制尚不清楚，从而阻止了优化材料的开发及其描述，从根本上讲是合理的模型。因此，拟议的研究具有三个主要目标： - 通过动态光散射（DLS）来确定葡聚糖移植矩阵的结构和动态行为，以了解这些系统中聚合物链的波动和结构动态。在这些材料中，使用单分子跟踪技术在纳米镜量表中确定单个蛋白质分子在用带电聚合物官能化的基质中的迁移率。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，并为实用速率预测构建物理上准确的概念和现象学模型。收录，这项研究的组成部分将提供尚未实现的对这些复杂运输现象的理解水平媒体是开发新的，更有效的材料的基础，并有助于设计和优化色谱过程。BOODER的影响：生物医学科学的进步提高了人们对基于生物学的新药物的预期，以预防，治疗和治愈严重的疾病，实际上，新的基于蛋白质的生物药物对人们的生活产生了巨大影响。但是，将这些生物分子分离和纯化的慢速扩散使它们变得复杂，这目前限制了工业生物色谱的性能。拟议的项目具有通过引入新的，更有效的固定阶段以及对运输现象的前所未有的理解水平来转移生物制药的下游处理的潜力和设计。研究和教育将通过将该项目的结果纳入大学级学生的教育模块，通过实验和分子建模中的协同活动加强研究生的教育，以及通过每年持有的蛋白质色谱专业人士的简短课程来加强研究生的教育在UVA。外展将包括开发实验室示范，以在高中生的上升计划中使用，并通过促进少数族裔本科生参加我们的短期课程，并通过招募人数不足的群体的毕业生。