Biochemical characterization of Wnt-Frizzled interactions using a soluble, biologically active vertebrate Wnt protein
Biochemical characterization of Wnt-Frizzled interactions using a soluble, biologically active vertebrate Wnt protein
复制标题
DOI：
10.1073/pnas.96.7.3546
复制
发表时间：
1999-03-30
影响因子：
11.1
通讯作者：
Nathans, J
中科院分区：
文献类型：
--
作者：
Hsieh, JC;Rattner, A;Nathans, J
Wnt 信号传导的生化研究因难以获得大量可溶性、具有生物活性的 Wnt 蛋白而受到阻碍。在本文中，我们报道了在果蝇 S2 细胞中产生具有生物活性的非洲爪蟾 Wnt8 (XWnt8)。表位或碱性磷酸酶标记的 XWnt8 蛋白由浓缩的 S2 细胞分泌，其形式适合定量生化实验，产量分别为 5 毫克/升和 0.5 毫克/升。还描述了在 293 细胞中产生小鼠 Frizzled 8 富含半胱氨酸结构域 (CRD) 的 IgG 融合体的条件，产量为 20 毫克/升。我们展示了使用这些蛋白质来研究可溶性 XWnt8 与各种卷曲蛋白、膜锚定或分泌的 CRD 以及果蝇卷曲 2 的 CRD 中的一组插入突变体之间的相互作用。在固相结合测定中，XWnt8-碱性磷酸酶融合体对纯化的小鼠卷曲 8-CRD-IgG 融合体的亲和力约为 9 纳米。
Biochemical studies of Wnt signaling have been hampered by difficulties in obtaining large quantities of soluble, biologically active Wnt proteins. In this paper, we report the production in Drosophila S2 cells of biologically active Xenopus Wnt8 (XWnt8). Epitope- or alkaline phosphatase-tagged XWnt8 proteins are secreted by concentrated S2 cells in a form that is suitable for quantitative biochemical experiments with yields of 5 and 0.5 mg per liter, respectively. Conditions also are described for the production in 293 cells of an IgG fusion of the cysteine-rich domain (CRD) of mouse Frizzled 8 with a yield of 20 mg/liter. We demonstrate the use of these proteins for studying the interactions between soluble XWnt8 and various Frizzled proteins, membrane anchored or secreted CRDs, and a set of insertion mutants in the CRD of Drosophila Frizzled 2. In a solid phase binding assay, the affinity of the XWnt8-alkaline phosphatase Fusion for the purified mouse Frizzled 8-CRD-IgG fusion is approximate to 9 nM.