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

该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运输来了解蛋白质层析，这在以前是不可能的。具体来说，这项工作将：(1)观察单个蛋白质分子在数千个吸附剂位点上的吸附和解吸，(2)确定每个位点上停留时间的分布，(3)直接观察位点的异质性，(4)测量激活能量，(5)测试配体密度和类型的影响，(6)表征不同表面亲和力但大小和形状相同的蛋白质之间的竞争，以及(7)测量单分子运输。提出的研究旨在通过使用单分子荧光，开辟一种全新的研究蛋白质色谱（和免疫测定，微阵列，生物传感器等）的方法。建立在联合pi的基础上？凭借我们在单分子光谱方面的经验，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（FCS）运输研究的方法。研究的特定要素包括测定单个蛋白质在单个吸附位点上的停留时间，这些停留时间的分布，以及配体密度、配体聚类、离子强度和竞争对手存在的影响。琼脂糖凝胶内部的传输行为将用FCS表征。这种方法将支持预测分子理论方法的发展，以模拟色谱过程。它将阐明簇电荷吸附剂优越性能的分子起源，并将对所有色谱分离基础上的竞争性蛋白质吸附和位移过程提供相当大的启发。拟议工作的更广泛影响应该是广泛的。生物分离，特别是色谱分离，在现代生物制药的制造成本和工艺复杂性中占主导地位，并在生物医学和生物技术研究中消耗了巨大的精力。在这一跨学科领域，训练有素的研究人员和流程开发人员感到短缺。簇状电荷吸附剂被认为是这项工作的一个组成部分，它有更广泛的应用前景。结果和方法应直接适用于核酸和生物偶联物的分离，以及HIC、IMAC和Protein A亲和等其他方法。这些方法也可以应用于非分离技术的研究，如免疫测定、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/纳米生物技术界面工作的良好培训机会。这些领域的就业都在快速增长，在可预见的未来，这些领域应该是一个非常富有成效的领域。休斯顿大学是美国最具种族多样性的城市研究型大学之一，参与这项研究的学生将反映出这种多样性。与教育相结合的机会是丰富的，在相关领域有多个REU和RET项目。

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.