Testing Feasibility of SlipChip Workflow for Membrane Protein Crystallization

测试膜蛋白结晶滑动芯片工作流程的可行性

• 批准号: 8057731
• 负责人: Liang Li
• 金额: $ 17.01万
• 依托单位: TALIS BIOMEDICAL CORPORATION
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8057731
• 关键词: Address; Area; Biological Assay; Chemicals; Complex; Crystallization; Detergents; Devices; Diffusion; Drug Delivery Systems; Enzymes; Equipment; Extravasation; Freezing; Glass; Goals; Health; Human; Immunoassay; In Situ; Knowledge; Lipids; Liquid substance; Membrane Proteins; Methods; Metric; Molds; Performance; Phase; Physiological Processes; Plastics; Play; Proteins; Protocols documentation; Public Health; Research; Resolution; Risk; Robotics; Role; Sampling; Screening procedure; Signal Transduction; Solutions; Structure; Surface Tension; Techniques; Technology; Testing; Time; Transducers; Viscosity; Work; X ray diffraction analysis; X-Ray Diffraction; biological research; commercialization; design; evaporation; innovation; innovative technologies; meetings; meter; miniaturize; nanolitre; receptor; scale up; three dimensional structure; tool

DESCRIPTION (provided by applicant): Membrane proteins are crucial to a wide variety of physiological processes, functioning as chemical receptors, transport channels and signal transducers. It is estimated that over half of drug targets are membrane proteins. Structural knowledge of membrane proteins is of very high biomedical significance. Crystallization of membrane proteins is an important approach to obtaining structural information of membrane proteins. However, obtaining diffracting crystals of membrane proteins is still difficult due to a number of bottlenecks, including low quantity and poor stability of membrane proteins; search of larger parameter space; and difficulty to obtaining diffraction sufficient to determine structures. No technology is currently available to address all of the needs and bottlenecks. SlipChip is an attractive and innovative technology that can potentially achieve these goals. It has been already applied to complex protocols such as enzyme assays, immunoassays, and PCR assays. The PI has demonstrated that SlipChip fabricated in glass can be used to handle and crystallize membrane proteins, and obtain crystals and high-resolution structures of soluble proteins. Yet, many technologies applicable to soluble proteins fail with membrane proteins; in addition, many elegant solutions to a single step in the crystallization workflow (e.g. establishing free interface diffusion) create additional bottlenecks elsewhere (e.g. creating problems in scale-up or crystal extraction). The goal of this Phase I proposal is to establish the feasibility and applicability of this innovation in the area of membrane proteins, while testing the feasibility of upstream and downstream steps in the crystallization workflow: from Aim 1) simple loading that will meter out nL volumes robustly for a range of solutions without the need for any equipment, to Aim 2) performance, from filling to incubation and observation, of chips made in plastic by inexpensive molding techniques, to Aim 3) robust methods of extraction and diffraction of crystals. Reaching these specific aims would firmly establish feasibility and viability of SlipChip technology for membrane protein crystallization, and would reduce the technical risk of Phase II work in this area, which would integrate many of the steps of concentration, separation, detergent exchange, purification, and formation of meso-phases into a single chip that accepts a fraction off a purification column and performs complex manipulations to produce diffraction-quality crystals of membrane proteins. PUBLIC HEALTH RELEVANCE: Structural knowledge of membrane proteins is of very high biomedical significance to public health. Crystallization of membrane proteins is an important approach to obtaining their structural information, but is difficult due to a number of bottlenecks. This proposal describes a SlipChip technology to address unmet needs and bottlenecks for membrane protein crystallization.

描述（由申请人提供）：膜蛋白对多种生理过程至关重要，作为化学受体、转运通道和信号转导器发挥作用。据估计，超过一半的药物靶标是膜蛋白。膜蛋白的结构知识具有很高的生物医学意义。膜蛋白的结晶是获取膜蛋白结构信息的重要途径。然而，获得膜蛋白的衍射晶体仍然是困难的，由于一些瓶颈，包括膜蛋白的低数量和差的稳定性;搜索更大的参数空间;以及难以获得足以确定结构的衍射。目前没有技术可以解决所有的需求和瓶颈。SlipChip是一种有吸引力的创新技术，可以实现这些目标。它已经应用于复杂的协议，如酶测定，免疫测定和PCR测定。PI已经证明，在玻璃中制造的SlipChip可用于处理和结晶膜蛋白，并获得可溶性蛋白质的晶体和高分辨率结构。然而，许多适用于可溶性蛋白质的技术在膜蛋白方面失败;此外，结晶工作流程中单个步骤的许多优雅解决方案（例如建立自由界面扩散）在其他地方产生了额外的瓶颈（例如在放大或晶体提取中产生问题）。该第一阶段提案的目标是确定该创新在膜蛋白领域的可行性和适用性，同时测试结晶工作流程中上游和下游步骤的可行性：从目标1）简单的装载，其将在不需要任何设备的情况下稳健地计量出nL体积，到目标2）性能，从填充到孵育和观察，通过廉价的模制技术在塑料中制造芯片，以实现目标3）晶体的提取和衍射的稳健方法。达到这些具体目标将牢固地建立用于膜蛋白结晶的滑动芯片技术的可行性和生存能力，并将降低该领域中的第二阶段工作的技术风险，该阶段工作将整合浓缩、分离、洗涤剂交换、纯化、纯化、纯化、分离、纯化和分离的许多步骤。以及中-这是一个简单的过程，它可以从纯化柱中分离出一部分，并进行复杂的操作，以产生衍射质量的膜蛋白晶体。 公共卫生相关性：膜蛋白的结构知识对公共卫生具有非常高的生物医学意义。膜蛋白的结晶是获得其结构信息的重要途径，但由于存在许多瓶颈而困难。该提案描述了滑动芯片技术，以解决膜蛋白结晶的未满足的需求和瓶颈。