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

1133965/1134417威尔逊/兰德斯这个项目的总体目标是通过观察单个蛋白质分子在现实吸附剂中的吸附和传输来了解蛋白质层析，这在以前是不可能的。具体地说，拟议的工作将：(1)观察单个蛋白质分子在数千个吸附中心上的吸附和解吸，(2)确定每个吸附中心的停留时间分布，(3)直接观察中心的异质性，(4)测量活化能，(5)测试配体密度和类型的影响，(6)表征表面亲和力不同但大小和形状相同的蛋白质之间的竞争，以及(7)测量单分子传输。这项拟议的研究旨在通过使用单分子荧光开辟一种全新的方法来研究蛋白质层析(以及免疫分析、微阵列、生物传感器等)。基于S在单分子光谱学方面的经验，以及我们之前在蛋白质单分子亲和识别方面的成功合作，我们开发了在现实的琼脂糖离子交换吸附剂中进行单分子成像和荧光相关光谱输运研究的方法。研究的具体内容包括单一蛋白质在单一吸附中心的停留时间的测定，这些停留时间的分布，以及配体密度、配体簇集、离子强度和竞争对手的存在的影响。琼脂糖凝胶内的传输行为将通过FCS来表征。这一方法将支持预测分子理论方法的发展，以模拟色谱过程。它将阐明簇状电荷吸附剂优异性能的分子来源，并将相当程度地阐明作为所有色谱分离基础的竞争性蛋白质吸附和置换过程。拟议工作的更广泛影响应该是广泛的。生物分离，特别是层析，主导了现代生物制药制造的成本和工艺复杂性，并消耗了生物医学和生物技术研究的巨大努力。在这一跨学科领域，训练有素的调查人员和流程开发人员明显不足。簇状电荷吸附剂将被描述为这项工作的一个元素，显示出更广泛的应用前景。结果和方法应直接适用于核酸和生物结合物的分离，以及其他方法，包括HIC，IMAC和蛋白A亲和。这些方法也可以应用于非分离技术的研究，如免疫分析、生物传感器和DNA微阵列。该项目将为学生提供在生物分离/生化技术和纳米生物学/纳米生物技术的界面上工作的极好的培训机会。这些领域中的每一个都享有快速的就业增长，在可预见的未来，接口应该是一个非常富有成效的领域。休斯顿大学是美国种族最多元化的城市研究型大学之一，参与这项研究的学生将反映这种多样性。与教育融合的机会很多，在相关领域有多个REU和RET计划。

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.