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)：在各种蛋白质组学倡议中，有大量工作是为了确定控制各种细胞过程的数千种蛋白质和蛋白质复合体的详细分子结构。用X射线结晶学和核磁共振进行蛋白质结构分析需要几十毫克的高纯度蛋白质。在过去的五年里，蛋白质结构倡议成功地开发了高通量的重组蛋白质结构测定方法，重点是所谓的“低挂果”，即在大肠杆菌中以可溶的、正确折叠的形式高水平表达的蛋白质，以及相对容易纯化和结晶的蛋白质。然而，许多对人类健康至关重要的靶标，如哺乳动物调节蛋白和膜蛋白，由于在这一过程中的一个或多个步骤存在困难，到目前为止一直被留在“高高在上的果实”后面。一个主要的障碍是，这些蛋白中的许多在大肠杆菌中表达水平很低和/或经历了异常折叠，因此需要使用真核系统，如昆虫细胞作为表达宿主。由于细胞提取物中非靶蛋白和干扰蛋白的比例较高，使用更复杂的表达系统为纯化带来了额外的挑战。在这些情况下，为细菌表达的蛋白质开发的亲和纯化方案无法产生结构分析所需的纯度，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，我们将测试采用模拟移动床(SMB)方法的概念，以通过固定化金属亲和层析(IMAC)来纯化多毫克规模的重组蛋白。在SMB方法中，固相在连续环路中相对于液体流动沿逆流方向运动。多个色谱池串联排列，进料和洗脱液连续输入，抽余液和洗脱液连续输出。最紧密结合的物种首先被释放，减少了结合物种的保留时间，并最小化了峰分散。通过调整缓冲液成分和流动参数，可以容易地优化纯化的目标物种的洗脱，从而使系统连续地将结合最强的物种从弱结合和非结合物种中分离出来。从历史上看，SMB层析几乎只应用于小分子异构体的大规模二元分离。我们将首先开发一个“迷你SMB”设备的原型，它克服了以前与缩小SMB设备相关的机械障碍，然后我们将测试该设备与标准方法在iMac纯化三种寡组氨酸标记的重组人在昆虫细胞中表达的方法。我们的项目将确定SMB层析的固有优势是否能够成功地应用于多毫克规模的高价值重组蛋白的IMAC纯化。 这项建议描述了一种新的设备和方法，它将有助于分离出足够纯度和数量的蛋白质，以进行可靠的结构分析。确定组成人类细胞的数千种蛋白质的详细原子结构将极大地提高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的治疗方法和药物，可以精确地针对特定的细胞途径和/或蛋白质，以更有效地进行疾病管理。