Collaborative Research: Ion-exchange adsorption of proteins: a single-molecule investigation

合作研究：蛋白质的离子交换吸附：单分子研究

• 批准号: 1134417
• 负责人: Christy Landes
• 金额: $ 20.42万
• 依托单位: William Marsh Rice University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1134417&HistoricalAwards=false
• 关键词: Collaborative; Research; Ion; exchange; adsorption

1133965/1134417Wilson/LandesThe overall goal of this project is to understand protein chromatography by observing the adsorption and transport of single protein molecules in realistic adsorbents, which has not previously been possible. Specifically, the proposed work will: (1) observe the adsorption and desorption of single protein molecules on thousands of adsorbent sites, (2) determine the distribution of dwell times at each site, (3) directly observe site heterogeneity, (4) measure activation energetics, (5) test the effects of ligand density and type, (6) characterize competition among proteins of different surface affinity but the same size and shape, and (7) measure single-molecule transport. The proposed research aims to open an entirely new way of investigating protein chromatography (and immunoassays, microarrays, biosensors, etc.), through the use of single-molecule fluorescence. Building on the co-PI?s experience in single-molecule spectroscopy, and our previous successful collaboration on single-molecule affinity recognition of proteins, we have developed methods for single-molecule imaging and fluorescence correlation spectroscopy (FCS) transport studies in realistic agarose ionexchange adsorbents. Particular elements of the investigation include the determination of the residence times of single proteins on single adsorbent sites, the distributions of these residence times, and the effects of ligand density, ligand clustering, ionic strength, and the presence of competitors. Transport behavior inside the agarose gel will be characterized by FCS. This approach will support the development of a predictive moleculartheoretic approach to modeling chromatographic processes. It will illuminate the molecular origins of the superior performance of clustered-charge adsorbents, and should shed considerable light on the competitive protein adsorption and displacement processes which underlie all chromatographic separations. The broader impacts of the proposed work should be extensive. Bioseparations, and chromatography in particular, dominate the cost and process complexity of manufacturing of modern biopharmaceuticals, and consume enormous effort in biomedical and biotechnological research. There is a felt shortage of trained investigators and process developers in this interdisciplinary area. The clustered-charge adsorbents to be characterized as an element of the work show promise for broader applications. The results and methods should be directly applicable to separations of nucleic acids and bioconjugates, and to other methods including HIC, IMAC, and Protein A affinity. These methods could also be applied to studies of non-separation technologies such as immunoassays, biosensors, and DNA microarrays. The project will provide excellent training opportunities for students to work at the interface of bioseparations/biochemical technology and nanobiology/nanobiotechnology. Each of these areas enjoys rapid employment growth, and the interface should be a very productive one for the foreseeable future. The University of Houston is one of the very most ethnically-diverse urban research universities in the United States, and the students involved in this research will reflect that diversity. Opportunities for integration with education are abundant, with multiple REU and RET programs in relevant areas.

1133965/1134417Wilson/Landesthe该项目的总体目标是通过观察现实的吸附剂中单蛋白分子的吸附和运输来了解蛋白质色谱法，以前尚不可能。 Specifically, the proposed work will: (1) observe the adsorption and desorption of single protein molecules on thousands of adsorbent sites, (2) determine the distribution of dwell times at each site, (3) directly observe site heterogeneity, (4) measure activation energetics, (5) test the effects of ligand density and type, (6) characterize competition among proteins of different surface affinity but the same size and shape, and （7）测量单分子运输。拟议的研究旨在通过使用单分子荧光来研究一种全新的研究蛋白质色谱（以及免疫测定，微阵列，生物传感器等）的方式。在Co-Pi在单分子光谱方面的经验以及我们以前在蛋白质单分子亲和力识别蛋白上的成功合作的基础上，我们开发了用于现实的琼脂糖琼脂糖离子化剂中的单分子成像和荧光相关光谱（FCS）传输研究的方法。研究的特定要素包括确定单个吸附剂位点上单蛋白的停留时间，这些停留时间的分布以及配体密度，配体簇，离子强度和竞争者的存在的影响。琼脂糖凝胶内部的运输行为将以FCS为特征。这种方法将支持建模色谱过程的预测分子理论方法的发展。它将阐明聚类 - 充电吸附剂的出色性能的分子起源，并应对竞争性蛋白质的吸附和位移过程揭示，这是所有色谱分离的基础。拟议的工作的更广泛影响应该是广泛的。尤其是生物序列，尤其是色谱法主导了现代生物制药制造的成本和过程复杂性，并在生物医学和生物技术研究中消耗了巨大的努力。在这个跨学科领域，训练有素的调查人员和过程开发人员感到缺乏。将其作为工作要素的聚类充值吸附剂显示出对更广泛应用的有望。结果和方法应直接适用于核酸和生物偶联物的分离，以及其他方法，包括HIC，IMAC和蛋白A亲和力。这些方法也可以应用于非分离技术的研究，例如免疫测定，生物传感器和DNA微阵列。该项目将为学生提供极好的培训机会，以在生物序列/生化技术和纳米生物学/纳米元素技术的界面工作。这些领域中的每一个都有快速的就业增长，在可预见的将来，界面应该是非常有生产力的。休斯顿大学是美国最多样化的城市研究所之一，参与这项研究的学生将反映出这种多样性。与教育融合的机会很丰富，在相关领域拥有多个REU和RET计划。