Novel Simulated Moving Bed Chromatography Device to Purify Recombinant Proteins

新型模拟移动床层析装置纯化重组蛋白

• 批准号: 7222029
• 负责人: ROBERT C MIERENDORF
• 金额: $ 10.77万
• 依托单位: SEMBA BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-15 至 2007-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7222029
• 关键词: Address; Affinity; Affinity Chromatography; Ally; Automation; Beds; Binding; Biology; Buffers; Cell Extracts; Cell physiology; Cells; Chimeric Proteins; Chromatography; Collection; Communicable Diseases; Complex; Condition; Crystallization; Crystallography; Development; Device Designs; Devices; Disease; Disease Management; Drug Industry; Eating; Elements; Enzymes; Escherichia coli; Food; Fruit; Genetic; Health; Human; Insecta; Isomerism; Laboratories; Lead; Left; Liquid substance; Manuals; Mechanics; Membrane Proteins; Metals; Methods; Molecular Structure; Operative Surgical Procedures; Output; Pathway interactions; Performance; Pharmacologic Substance; Phase; Phosphotransferases; Plant Resins; Positioning Attribute; Process; Protein Structure Initiative; Proteins; Proteomics; Protocols documentation; Rate; Reading; Recombinant Proteins; Recombinants; Refractory; Relative (related person); Resolution; Running; Sampling; Scheme; Series; Simulate; Solid; Solubility; Standards of Weights and Measures; Stream; Structural Protein; Structure; System; Tertiary Protein Structure; Testing; Time; Work; X-Ray Crystallography; base; computer program; concept; cost; countercurrent chromatography; feeding; fluid flow; improved; innovation; instrumentation; interest; milligram; novel; novel therapeutics; numb protein; pressure; protein expression; protein function; protein purification; protein structure; prototype; seal; small molecule; structural biology; three dimensional structure

DESCRIPTION (provided by applicant): In various proteomics initiatives there is a significant effort to determine detailed molecular structures of the thousands of proteins and protein complexes that govern various cellular processes. Protein structural analysis by X-ray crystallography and NMR requires tens of milligrams of highly purified proteins. Over the past five years, the Protein Structure Initiative has been successful in developing high-throughput methods for structural determination of recombinant proteins, focusing on the so-called "low-hanging fruit", i.e. proteins which are expressed at high levels as soluble, correctly folded species in E. coli, and which are relatively easily purified and crystallized. However, many targets important for human health, such as mammalian regulatory and membrane proteins, have thus far been left behind as "high-hanging fruit" due to difficulties with one or more steps in the process. One major hurdle is the fact that many of these proteins are poorly expressed and/or undergo aberrant folding in E. coli, necessitating the use of eukaryotic systems, such as insect cells, as the expression host. The use of more complex expression systems introduces additional challenges for purification due to the higher proportion of non-target and interfering proteins in cellular extracts. In these cases the affinity purification schemes developed for bacterial-expressed proteins fail to produce the purity required for structural analysis, necessitating additional purification steps which are expensive and difficult to automate. To address this problem, we will test the concept of adapting a simulated moving bed (SMB) approach to the multi- milligram scale purification of recombinant proteins by immobilized metal affinity chromatography (IMAC). In the SMB method, the solid phase moves in a countercurrent direction relative to the liquid flow in a continuous loop. Multiple chromatographic cells are arranged in a series with continuous input of feed and eluant streams and continuous output of raffinate and eluate streams. The most tightly bound species are released first, reducing retention time of bound species and minimizing peak dispersion. Elution of purified target species can be easily optimized by adjustment of buffer composition and flow parameters, such that the system continuously resolves the strongest binding species from weakly- and non-binding species. Historically, SMB chromatography has been almost exclusively applied to large-scale binary separations of small molecule isomers. We will first develop a prototype "mini-SMB" device that overcomes previous mechanical barriers related to scaled-down SMB devices, and then we will test the device vs. standard methods in IMAC purification of three oligohistidine-tagged recombinant human kinases expressed in insect cells. Our project will determine if the inherent advantages of SMB chromatography can be successfully applied to IMAC purification of high-value recombinant proteins at the multi-milligram scale. This proposal describes a novel device and method that would facilitate the isolation of proteins of sufficient purity and quantity for reliable structural analysis. The determination of detailed atomic structures of the thousands of proteins that comprise human cells will greatly increase our understanding of the fundamental mechanisms of normal and disease states. Such information will lead to the discovery of new therapies and pharmaceuticals that can be precisely targeted to specific cellular pathways and/or proteins for more effective disease management.

描述（由申请人提供）：在各种蛋白质组学计划中，要确定支配各种细胞过程的数千种蛋白质和蛋白质复合物的详细分子结构。 X射线晶体学和NMR的蛋白质结构分析需要数十毫克高度纯化的蛋白质。在过去的五年中，蛋白质结构计划成功地开发了用于确定重组蛋白质结构的高通量方法，重点是所谓的“低悬挂果实”，即以高水平表达的蛋白质，作为可溶性，正确折叠的coli中的coli中的蛋白质，并相对易于纯化和结晶。但是，许多对人类健康至关重要的目标，例如哺乳动物调节和膜蛋白，迄今为止，由于在此过程中有一个或多个步骤的困难，迄今为止，由于遇到或多个步骤而被抛弃为“高刺果”。一个主要的障碍是，这些蛋白质中的许多表达不佳和/或在大肠杆菌中经历异常折叠，因此需要使用真核系统（例如昆虫细胞）作为表达宿主。更复杂的表达系统的使用引入了纯化的额外挑战，这是由于细胞提取物中的非目标和干扰蛋白的比例较高。在这些情况下，针对细菌表达的蛋白质开发的亲和力纯化方案无法产生结构分析所需的纯度，因此需要额外的纯化步骤，这些步骤昂贵且难以自动化。为了解决这个问题，我们将测试通过固定的金属亲和力色谱法（IMAC）对重组蛋白进行多毫克量表纯化的模拟移动床（SMB）方法的概念。在SMB方法中，相对于连续循环中液体流动，固相移动朝着反电流的方向移动。多个色谱细胞以连续输入和洗脱流的连续输入以及拉咖啡酸盐和洗脱流的连续输出进行排列。最紧密的物种首先释放，从而减少了结合物种的保留时间，并最大程度地减少了峰分散体。可以通过调整缓冲液组成和流程参数来轻松优化纯化的靶物质物种的洗脱，从而使系统连续解析从弱和非结合物种中的最强结合物种。从历史上看，SMB色谱法几乎完全应用于小分子异构体的大规模二元分离。我们将首先开发一种原型“ Mini-SMB”设备，该设备可以克服与缩小的SMB设备相关的先前机械屏障，然后我们将测试设备与标准方法在iMAC纯化昆虫细胞中表达的三种寡聚性重组的人类激酶。我们的项目将确定SMB色谱法的固有优势是否可以成功应用于在多毫克量表上高价值重组蛋白的iMAC纯化。 该提案描述了一种新颖的装置和方法，该方法将促进具有足够纯度和数量的蛋白质分离以进行可靠的结构分析。确定构成人类细胞的数千种蛋白质的详细原子结构将大大增加我们对正常和疾病状态的基本机制的理解。这些信息将导致发现新疗法和药物，这些疗法和药物可以精确针对特定的细胞途径和/或蛋白质，以实现更有效的疾病管理。