Molecular Engineering of Multiple Weak Interactions for High Selectivity Bioseparations

高选择性生物分离的多重弱相互作用的分子工程

• 批准号: 1160039
• 负责人: Steven Cramer
• 金额: $ 34万
• 依托单位: Rensselaer Polytechnic Institute
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-15 至 2017-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1160039&HistoricalAwards=false
• 关键词: Molecular; Engineering; Multiple; Weak; Interactions

1160039CramerThis work will employ several state of the art libraries of novel ligands and materials in concert with protein libraries in a detailed study of MM ligand--protein interactions in the presence of FPMs. High throughput batch and column experiments will be performed using several protein libraries to screen for selectivity trends based on the different interaction moieties present on the ligands and modifiers. Protein pairs and MM ligand/FPM combinations exhibiting unique selectivities will then be investigated using NMR and MD simulations. Solution NMR titration experiments will be carried out with labeled proteins and MM ligands to determine changes in binding kinetics, association affinities, and ligand binding sites on the proteins in the presence of FPMs. Hyrodgen-deuterium exchange experiments and SS-NMR will be performed with resin bound proteins to examine protein-ligand binding interactions in solid phase systems. Extensive MD simulations will be performed on increasingly complex systems to develop a new framework for the understanding, manipulation, and prediction of multiple weak interactions in MM systems in the presence of FPMs. This will be achieved by (i) developing a database of the effects of FPMs on the fundamental water-mediated interactions using model solutes; (ii) identifying the key contributors to MM ligand-protein interactions and their synergies; (iii) developing tools to predict the effects of FPMs on protein-ligand MM interactions and carrying out MD simulations to provide insights into the importance of ligand flexibility, steric effects, and avidity effects. The knowledge base created by the experimental and theoretical studies will then be used to formulate a process for designing MM separations and validation of process will be carried out using Fab variants. Finally, the refined process will be applied to engineer efficient separation conditions for the purification of complex industrial mixtures provided by our industrial collaborators The proposed project will provide a fundamental understanding of the nature of MM ligand binding to proteins in both solution and solid systems in the presence of FPMs which will have a significant impact on the development and implementation of MM chromatographic technology for downstream purification. This work will enable the design of next generation MM chromatographic systems which will have important implications for separations ranging from large scale biopharmaceuticals to complex bioanalytical applications. Since the balance of weak interactions is the norm and not the exception in condensed high dielectric media, the systematic studies described in this proposal have the potential to provide new understanding of this important problem which will be readily translated to many related biological (molecular recognition, binding, aggregation), bioprocessing (formulation) and biomedical systems (bone regeneration, biomaterial design). The proposed research will also have an important impact on the education of both graduate and undergraduate chemical engineering students. The Cramer laboratory has a long track record of producing chemical engineers with a first rate training in chromatographic bioprocessing. This training will continue with the graduate students and undergraduates involved in this project. The proposed research will be carried out in the Center for Biotechnology and Interdisciplinary Studies which provides excellent multidisciplinary training opportunities for graduate and undergraduate students. Students will gain exposure to cell culture, solution NMR spectroscopy, chromatography and molecular simulations. The research developed in this project will also be incorporated into a chemical engineering senior laboratory chromatography experiment recently developed the PI as well as a course on Chromatographic Separation Processes. Finally, simulations performed in this project, as well as conceptual parts of molecular interactions will motivate new aspects of the Molecularium project, which uses animation movies to teach and inspire students at all levels about the fascinating world of molecules.

1160039 Cramer这项工作将采用几种最新的配体和材料的库，与蛋白质库一起详细研究在FPM存在下MM配体-蛋白质相互作用。 将使用几种蛋白质文库进行高通量批次和柱实验，以基于配体和修饰剂上存在的不同相互作用部分筛选选择性趋势。蛋白质对和MM配体/FPM组合表现出独特的选择性，然后将使用NMR和MD模拟进行研究。将使用标记的蛋白质和MM配体进行溶液NMR滴定实验，以确定在存在FPM的情况下蛋白质上的结合动力学、缔合亲和力和配体结合位点的变化。将使用树脂结合蛋白进行氢-氘交换实验和SS-NMR，以检查固相系统中的蛋白-配体结合相互作用。广泛的MD模拟将在日益复杂的系统上进行，以开发一个新的框架，用于理解，操纵和预测在FPMS存在下MM系统中的多个弱相互作用。这将通过（i）使用模型溶质开发FPM对基本水介导的相互作用的影响的数据库来实现;（ii）确定MM配体-蛋白质相互作用及其协同作用的关键因素;（iii）开发预测FPM对蛋白质-配体MM相互作用的影响的工具，并进行MD模拟以提供对配体柔性、空间效应、和亲合力效应。通过实验和理论研究创建的知识库将用于制定设计MM分离的工艺，并将使用Fab变体进行工艺验证。最后，该精制工艺将应用于设计有效的分离条件，用于纯化我们的工业合作者提供的复杂工业混合物。拟议的项目将提供对在FPMs存在下溶液和固体系统中MM配体与蛋白质结合的性质的基本理解，这将对MM色谱技术的开发和实施产生重大影响，下游纯化。这项工作将使下一代MM色谱系统的设计，这将有重要的意义，从大规模的生物制药分离复杂的生物分析应用。由于弱相互作用的平衡是常态，而不是例外，在凝聚的高介电介质中，本提案中描述的系统研究有可能提供对这一重要问题的新理解，这将很容易转化为许多相关的生物（分子识别，结合，聚集），生物加工（配方）和生物医学系统（骨再生，生物材料设计）。建议的研究也将有一个重要的影响，研究生和本科化学工程专业的学生的教育。Cramer实验室在培养具有一流色谱生物处理培训的化学工程师方面有着悠久的历史。将继续对参与该项目的研究生和本科生进行培训。拟议的研究将在生物技术和跨学科研究中心进行，该中心为研究生和本科生提供了极好的多学科培训机会。学生将接触到细胞培养，溶液核磁共振光谱，色谱和分子模拟。本计画之研究也将纳入PI最近开发的化学工程高级实验室层析实验以及层析分离过程课程。最后，在这个项目中进行的模拟，以及分子相互作用的概念部分将激发Molecularium项目的新方面，该项目使用动画电影来教授和激励各级学生了解迷人的分子世界。