CRYSTAL STRUCTURE OF THE ENTIRE EXTRACELLULAR DOMAIN OF C-KIT RECEPTOR (THE STEM

C-KIT 受体整个胞外域的晶体结构（茎）

• 批准号: 7726203
• 负责人: JOSEPH SCHLESSINGER
• 金额: $ 1.01万
• 依托单位: BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-18 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7726203
• 关键词: Anions; Binding; Biological; Bioreactors; Cell Cycle; Cell Differentiation process; Cell Proliferation; Cell Survival; Cell physiology; Cells; Chromatography; Computer Retrieval of Information on Scientific Projects Database; Condition; Crystallization; Data; Data Set; Dimerization; Enzymes; Extracellular Domain; Freezing; Funding; Grant; Home environment; Insecta; Institution; Learning; Ligands; Mediating; Metabolism; Metals; Play; Procedures; Protein Kinase; Proteins; Proto-Oncogene Protein c-kit; Protocols documentation; Receptor Protein-Tyrosine Kinases; Research; Research Personnel; Resources; Role; Screening procedure; Shapes; Signal Transduction; Source; Spottings; Stem Cell Factor; Structure; Tissues; United States National Institutes of Health; Work; cell dimension; cell motility; improved; receptor; size; stem

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cell signaling by receptor tyrosine kinases (RTKs) plays a pivotal role in the control of many cellular processes including the cell cycle, cell migration, cell metabolism, cell survival, cell proliferation and cell differentiation. The c-Kit receptor is an RTK that is expressed in many tissues and mediates its biological effects following interactions with its ligand, stem cell factor (SCF). SCF binding leads to c-Kit dimerization, stimulation of autophosphorylation and activation of the intrinsic protein kinase activity. Although much work has been devoted to analysis of c-Kit signaling, important questions of mechanism regarding how the receptor avoids dimerization and activation in the absence of the SCF, remain unanswered. In order to learn more about the auto inhibition and activation mechanisms, we propose to solve the structure of the extracellular domain of c-Kit in its unoccupied form. The following expression and purification procedure is considerably improved in comparison to previous protocol. The entire extracellular domain of c-Kit containing five Ig-domains is expressed in Sf9 insect cells using wave bioreactor. The expression yield from 15 litter culture is 30 mg of crude glycosylated protein after metal-chelating chromatography. Following deglycosylation treatment using Endoglycosydase F1 enzyme and anion exchange chromatography, 15 mg of a homogeneous (estimated by SDS and Native-PAGE) deglycasylated protein is concentrated and frozen. After extensive screening (600 conditions for each form), crystallization conditions were found for unoccupied deglycosylated c-Kit receptor. The initial crystallization conditions for the unoccupied c-Kit are 10% polyethyleneglycol 1000 at pH 6.0. After its crystallization conditions were optimized, crystal shapes and sizes are improved with size varies from 75-150 microns. Using home-source diffractometer (Rigaku R-Axis 4), with 90 minutes exposure, the crystals diffract up to 8 A and spots can be detected upto 5 A. Recently we collected data on the NSLS x29 beam line. We got couple of native data sets upto 3.0 A with 96% completeness. The crystals belong to the rhombohedral space group R3, with unit-cell dimensions a = b = 162, c = 66 A.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 受体酪氨酸激酶（RTK）的细胞信号在控制许多细胞过程中起关键作用，包括细胞周期，细胞迁移，细胞代谢，细胞存活，细胞增殖和细胞分化。 C-KIT受体是在许多组织中表达的RTK，并在与配体的干细胞因子（SCF）相互作用后介导其生物学作用。 SCF结合导致C-KIT二聚化，自磷酸化的刺激和固有蛋白激酶活性的激活。 尽管已经致力于分析C-KIT信号传导，但有关受体如何在没有SCF的情况下避免二聚化和激活机制的重要问题仍未得到答复。 为了了解有关自动抑制和激活机制的更多信息，我们建议以其无占用形式解决C-KIT细胞外域的结构。 与先前的协议相比，以下表达和纯化程序得到了显着改进。 使用波生物反应器在SF9昆虫细胞中表达含有五个Ig域的C-KIT的整个细胞外域。 15个垃圾培养的表达产量为金属螯合色谱后30 mg粗糖基化蛋白。使用内胚酶酶F1酶和阴离子交换色谱法进行脱糖基化处理后，将15 mg的均质（通过SDS和天然页面估算）浓缩并冷冻蛋白。在广泛的筛选（每种形式的600条条件）之后，发现无置脱糖基化的C-KWIT受体的结晶条件。在pH 6.0时，未占用的C-KIT的初始结晶条件为10％聚乙烯1000。 优化其结晶条件后，晶体形状和尺寸改善，大小从75-150微米变化。使用家庭源衍射仪（Rigaku r轴4），暴露90分钟，晶体衍射量最大为8 a，可检测到最高5的斑点。 最近，我们收集了NSLS X29梁线上的数据。我们的本机数据设置为3.0 A，完整性为96％。 晶体属于菱形空间群R3，其单位细胞尺寸a = b = 162，c = 66A。