This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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.
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