Hoffmann Anita, Neumann Piotr, Schierhorn Angelika, Stubbs Milton T
Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Abteilung Physikalische Biotechnologie, Kurt-Mothes-Strasse 3, 06120 Halle (Saale), Germany.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Aug 1;64(Pt 8):707-10. doi: 10.1107/S1744309108018812. Epub 2008 Jul 5.
The Spätzle protein is involved in both the definition of the dorsal-ventral axis during embryonic development and in the adult innate immune response. The disulfide-linked dimeric cystine-knot protein has been expressed as a proprotein in inclusion bodies in Escherichia coli and refolded in vitro by rapid dilution. Initial orthorhombic crystals that diffracted to 7 A resolution were obtained after three months by the sitting-drop vapour-diffusion method. Optimization of the crystallization conditions resulted in orthorhombic crystals (space group P2(1)2(1)2(1), with unit-cell parameters a = 53.0, b = 59.2, c = 62.5 A) that diffracted to 2.8 A resolution in-house. The small volume of the asymmetric unit indicated that it was not possible for the crystals to contain the complete pro-Spätzle dimer. Mass spectrometry, N-terminal sequencing and Western-blot analysis revealed that the crystals contained the C-terminal disulfide-linked cystine-knot dimer. Comparison of various crystallization experiments indicated that degradation of the N-terminal prodomain was dependent on the buffer conditions.
斯帕兹勒蛋白在胚胎发育过程中的背腹轴定义以及成年个体的固有免疫反应中均发挥作用。这种二硫键连接的二聚体胱氨酸结蛋白在大肠杆菌的包涵体中以前体蛋白形式表达，并通过快速稀释在体外进行重折叠。采用坐滴气相扩散法，三个月后获得了最初衍射分辨率为7埃的正交晶体。对结晶条件进行优化后得到了正交晶体（空间群P2(1)2(1)2(1)，晶胞参数a = 53.0、b = 59.2、c = 62.5埃），其在内部衍射分辨率达到2.8埃。不对称单元的体积较小，这表明晶体不可能包含完整的前体斯帕兹勒二聚体。质谱分析、N端测序和蛋白质免疫印迹分析表明，晶体包含C端二硫键连接的胱氨酸结二聚体。各种结晶实验的比较表明，N端前结构域的降解取决于缓冲液条件。
