Chemically Selective Displacers for Protein Purification

建议编号：0730830 PRINCIPAL调查员：Steven CramerINSTUTION：伦斯勒理工学院PROPOSAL标题：用于蛋白质纯化的化学选择性置换剂将使用自动化液体处理系统检测蛋白质、置换剂结构、流动相特性和置换剂浓度对置换层析系统的效率和选择性的影响，以实现多维筛选。从高通量筛选(HTS)研究中选择的高亲和力和选择性置换先导化合物将被合成。在整个合成工作中，将研究将生色团和/或荧光团引入这些置换剂的各种方法，以便于在线检测。这项工作将研究具有与特定蛋白质类结合能力的置换体。除了HTS研究确定的选择性置换剂外，这项工作还将研究使用已知结合配体连接到适当带电部分的类选择性置换剂的先验设计。在该项目的这一阶段开发的置换剂将在批处理和柱式实验中评估其选择性置换所需蛋白质的能力。还将使用多种技术研究选择性置换剂和蛋白质之间的相互作用模式。基于空间质量作用等温线的四态模拟模型将被用来预测这些选择性置换剂的柱性能。拟议的研究将对大规模生物处理以及分析生物技术产生影响。开发与特定蛋白质或蛋白质类别结合的选择性置换剂，同时作为有效的置换剂，将极大地提高离子交换系统的选择性。这项拟议的工作可能会对参与细胞调控的关键蛋白质的纯化产生重大影响。这项拟议的研究也将对化学工程研究生和本科生的教育产生重要影响。参与这个多学科项目的学生将在色谱生物处理、建模、机器人和各种分析技术(如毛细管区带电泳、表面等离子体共振和核磁共振)方面获得重要培训。这个项目的研究将被纳入一门关于色谱分离过程的课程。最后，在PI实验室实习的高中生也将获得重要的实验室经验。如果成功，该提案将对生物加工和一些与健康有关的领域产生重大而广泛的影响。

PROPOSAL NUMBER: 0730830PRINCIPAL INVESTIGATOR: Steven CramerINSTITUTION: Rensselaer Polytechnic InstitutePROPOSAL TITLE: Chemically Selective Displacers for Protein PurificationThe effects of protein, displacer structure, mobile phase characteristics and displacer concentration on the efficacy and selectivity of displacement chromatography systems will be examined using an automated liquid handling system to enable multi-dimensional screens. High affinity and selective displacer lead compounds selected from the high throughput screening (HTS) studies will be synthesized. Throughout this synthesis work various approaches to introduce chromophores and/or fluorophores into these displacers will be examined in order to facilitate on-line detection. This work will examine displacers that have the ability to bind to specific classes of proteins. In addition to selective displacers identified from the HTS studies, this work will investigate the a priori design of class selective displacers using known binding ligands tethered to appropriately charged moieties. Displacers developed in this phase of the project will be evaluated for their ability to selectively displace the desired proteins under batch and column experiments. The mode of interaction between selective displacers and proteins will also be investigated using multiple techniques. A four state simulation model based on the steric mass action isotherm will be used to predict column performance of these selective displacers. The proposed research will have an impact on both large scale bioprocessing as well as analytical biotechnology. The development of selective displacers that bind to a specific protein or protein class while also acting as efficient displacers will dramatically increase the selectivity of ion exchange systems. The proposed work could have a major impact on the purification of key proteins involved in cellular regulation. The proposed research will also have an important impact on the education of both graduate and undergraduate chemical engineering students. The students involved in this multidisciplinary project will obtain significant training in chromatographic bioprocessing, modeling, robotics, and a variety of analytical techiques such as capillary zone electrophoresis, surface plasmon resonance and NMR. The research from this project will be incorporated into a course on Chromatographic Separation Processes. Finally, high school students who are carrying out internships in the PI's laboratory will also obtain important laboratory experience. If successful the proposal will have a significant and broad impact on bioprocessing and a number of health related fields.