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

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。受体酪氨酸激酶(RTK)介导的细胞信号转导在细胞周期、细胞迁移、细胞代谢、细胞存活、细胞增殖和细胞分化等多种细胞过程中起着重要的调控作用。C-Kit受体是一种RTK，在许多组织中表达，并与其配体干细胞因子(SCF)相互作用，介导其生物学效应。SCF结合导致c-Kit二聚化，刺激自身磷酸化，激活固有的蛋白激酶活性。尽管对c-Kit信号的分析已经投入了大量的工作，但关于受体如何在没有SCF的情况下避免二聚化和激活的重要机制问题仍未得到解答。为了更多地了解c-Kit的自抑制和激活机制，我们建议解决c-Kit胞外结构域的空位形式。与以前的方法相比，下面的表达和纯化步骤有了很大的改进。利用WAVE生物反应器在Sf9昆虫细胞中表达了含有5个Ig结构域的c-Kit的完整胞外区。经金属螯合层析后，15窝培养的表达产物为30 mg粗糖化蛋白。在使用内切酶F1酶和阴离子交换层析进行脱糖处理后，浓缩和冷冻15毫克均一的(经SDS和Native-PAGE鉴定的)脱糖蛋白。经过广泛的筛选(每种形式600个条件)，找到了未被占据的去糖基化c-Kit受体的结晶条件。空白c-Kit的初始结晶条件为10%的聚乙二醇1000，pH为6.0。晶化条件优化后，晶体的形状和尺寸都得到了改善，尺寸在75-150微米之间。使用主源衍射仪(Rigaku R-Axis 4)，曝光90分钟，晶体的衍射高达8A，光斑可以探测到高达5A。最近，我们收集了NSLS x29光束线上的数据。我们得到了两个高达3.0A的原生数据集，完备率为96%。晶体属于菱面体空间群R3，晶胞尺寸a=b=162，c=66A。

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.