Windsor W T, Walter L J, Syto R, Fossetta J, Cook W J, Nagabhushan T L, Walter M R
Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.
Proteins. 1996 Sep;26(1):108-14. doi: 10.1002/(SICI)1097-0134(199609)26:1<108::AID-PROT10>3.0.CO;2-K.
X-ray diffraction quality crystals have been obtained from a complex between interferon gamma and the extracellular domain of its high-affinity cell surface receptor. The crystals were obtained from interferon gamma/interferon gamma receptor complexes purified by size exclusion chromatography. Diffraction quality crystals required analyzing these complex samples by isoelectric focusing gels to select purified complex fractions devoid of unbound interferon gamma. These studies used interferon gamma receptor engineered with an eight amino acid N-terminal deletion to eliminate heterogeneity generated due to proteolytic cleavage. In addition, the receptor was expressed in an E. coli secretion cell line which eliminated the need to refold the protein. Hexagonal crystals were grown from 1.6 M ammonium phosphate solutions and belong to a spacegroup of P6(5)22 with unit cell dimensions a = 145.9 A and c = 180.3 A. These crystals diffract to at least 2.9 A resolution when exposed to synchrotron radiation. SDS PAGE analysis of the crystals demonstrated that both interferon gamma and the receptor were present. Analysis of the x-ray diffraction data revealed that the crystals contain complexes with a stoichiometry of 2:1 receptor: ligand within the crystallographic asymmetric unit and consist of approximately 55% solvent.
已从干扰素γ与其高亲和力细胞表面受体的细胞外结构域之间的复合物中获得了X射线衍射质量的晶体。这些晶体是从通过尺寸排阻色谱法纯化的干扰素γ/干扰素γ受体复合物中获得的。获得衍射质量的晶体需要通过等电聚焦凝胶分析这些复杂样品，以选择不含未结合干扰素γ的纯化复合物级分。这些研究使用了经工程改造的干扰素γ受体，该受体N端缺失八个氨基酸，以消除由于蛋白水解切割产生的异质性。此外，该受体在大肠杆菌分泌细胞系中表达，从而无需对蛋白质进行重折叠。从1.6 M磷酸铵溶液中生长出六方晶体，属于空间群P6(5)22，晶胞尺寸a = 145.9 Å，c = 180.3 Å。当暴露于同步辐射时，这些晶体的衍射分辨率至少为2.9 Å。对晶体的SDS-PAGE分析表明，干扰素γ和受体均存在。对X射线衍射数据的分析表明，晶体在晶体学不对称单元中包含化学计量比为2:1受体:配体的复合物，并且由约55%的溶剂组成。
