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，人们付出了巨大的努力来确定控制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。通过 X 射线晶体学和 NMR 进行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，蛋白质结构计划已成功开发了重组蛋白结构测定的高通量方法，重点关注所谓的“容易实现的目标”，即以可溶性、高水平表达的蛋白质。大肠杆菌中正确折叠的物种，并且相对容易纯化和结晶。然而，许多对人类健康重要的靶标，例如哺乳动物调节蛋白和膜蛋白，由于这一过程中一个或多个步骤的困难，迄今为止一直被视为“悬而未决的成果”。一个主要障碍是许多这些蛋白质在大肠杆菌中表达不良和/或发生异常折叠，因此需要使用真核系统（例如昆虫细胞）作为表达宿主。由于细胞提取物中非目标蛋白和干扰蛋白的比例较高，使用更复杂的表达系统给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）方法通过固定化金属亲和层析（IMAC）进行多毫克规模重组蛋白纯化的概念。在 SMB 方法中，固相在连续回路中相对于液流沿逆流方向移动。多个色谱单元串联排列，连续输入进料和洗脱液流，连续输出萃余液和洗脱液流。最紧密结合的物质首先被释放，减少了结合物质的保留时间并最大限度地减少了峰分散。通过调整缓冲液成分和流动参数，可以轻松优化纯化目标物质的洗脱，从而使系统不断地从弱结合物质和非结合物质中分离出最强的结合物质。从历史上看，SMB 色谱法几乎专门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原型“mini-SMB”设备，该设备克服了之前与按比例缩小的 SMB 设备相关的机械障碍，然后我们将在昆虫中表达的三种寡聚组氨酸标记的重组人激酶的 IMAC 纯化中测试该设备与标准方法的比较细胞。我们的项目将确定 SMB 层析的固有优势是否可以成功应用于多毫克规模的高价值重组蛋白的 IMAC 纯化。 该提案描述了一种新颖的设备和方法，有助于分离足够纯度和数量的蛋白质，以进行可靠的结构分析。确定构成人体细胞的数千种蛋白质的详细原子结构将极大地增进我们对正常和疾病状态基本机制的理解。这些信息将导致新疗法和药物的发现，这些疗法和药物可以精确地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。