Development of Biosensor Technology for Protein Interactions

由于地表粗糙，地表的物理和化学微环境是不均匀的。 因此，在溶液中具有单一类结合位点的蛋白质一旦化学交联到表面，就可以显示结合性质的分散。 作为一种工具来研究这个合奏的表面网站，我们以前已经介绍了一种计算方法来确定分布的亲和力和动力学结合位点参数的实验表面结合数据。 这使我们能够，第一次，同时考虑到两个最常见的实验问题的表面结合时，使用生物传感器表征蛋白质的相互作用，网站的异质性和质量传输的限制，从而模型实验数据一致的数据采集的噪声水平内。 这充分利用了表面等离子体共振生物传感器的高灵敏度的蛋白质相互作用的研究，并提供了前所未有的详细程度的表面结合位点的信息。 这种方法的进一步应用，以更多的蛋白质相互作用，巩固了我们的知识的表面位点的异质性。 我们已经开发了一个独立的软件，允许其他科学家导入实验表面结合动力学数据集和计算网站的功能分布。 我们已经用模型蛋白质系统证实了直接蛋白质固定化如何导致异质性和具有不同亲和力的亚群。 我们已经实现了用于常规分析的软件，其具有定量溶液中蛋白质相互作用的溶液竞争测定。

Due to surface rugosity, the physical and chemical microenvironment at the surface is heterogeneous. Therefore, proteins that have a single class of binding sites in solution can show a dispersion in the binding properties once chemically crosslinked to the surface. As a tool to study this ensemble of surface sites, we have previously introduced a computational approach to determine distributions of affinity and kinetic binding site parameters from experimental surface binding data. This allowed us, for the first time, to account simultaneously for the two most commonly encountered experimental problems of surface binding when using biosensors for characterizing protein interactions, site heterogeneity and mass transport limitation, and thereby model experimental data consistently to within the level of noise of data acquisition. This fully exploits the high sensitivity of surface plasmon resonance biosensors for the study of protein interactions, and provides information of the surface binding sites with unprecedented level of detail. Further applications of this approach to more protein interactions have solidified our knowledge of surface site heterogeneity. We have developed a stand-alone software that allows other scientist to import experimental surface binding kinetics data sets and compute the functional distribution of sites. We have confirmed with a model protein system how the direct protein immobilization can cause heterogeneity and sub-populations with different affinity. We have implemented software for routine analysis with a solution competition assay that quantifies protein interactions in solution.