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

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 通过受体酪氨酸激酶（RTK）的细胞信号传导在许多细胞过程的控制中起关键作用，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受体是一种RTK，在许多组织中表达，并在与其配体干细胞因子（SCF）相互作用后介导其生物学效应。SCF结合导致c-Kit二聚化，刺激自磷酸化和激活内在蛋白激酶活性。 虽然大量的工作已经致力于分析c-Kit信号，重要的问题的机制，如何避免二聚化和激活的SCF的情况下，受体，仍然没有答案。 为了了解更多关于自动抑制和激活机制，我们建议解决c-Kit的胞外结构域的结构在其未被占用的形式。 与先前的方案相比，以下表达和纯化程序得到显著改进。 利用wave生物反应器在Sf9昆虫细胞中表达含有5个Ig结构域的c-Kit的整个胞外结构域。在金属螯合层析后，来自15窝培养物的表达产量为30mg粗糖基化蛋白。使用内切糖基化酶F1酶和阴离子交换色谱法进行去糖基化处理后，浓缩并冷冻15 mg均质（通过SDS和Native-PAGE估计）去糖基化蛋白。经过广泛筛选（每种形式600种条件），找到了未占据的去糖基化c-Kit受体的结晶条件。未被占据的c-Kit的初始结晶条件为10%聚乙二醇1000，pH 6.0。 通过优化晶化条件，晶体形状和尺寸得到改善，尺寸在75 - 150 μ m之间。使用家庭源衍射仪（Rigaku R-Axis 4），曝光90分钟，晶体的衍射强度高达8 A，斑点可检测到高达5 A。 最近，我们收集了NSLS x29光束线的数据。我们得到了几个高达3.0 A的本地数据集，完整性为96%。 晶体属于菱面体空间群R3，晶胞尺寸a = B = 162，c = 66。

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.