利用可溶性、具有生物活性的脊椎动物Wnt蛋白对Wnt-卷曲蛋白相互作用进行生化特性分析。
Biochemical characterization of Wnt-frizzled interactions using a soluble, biologically active vertebrate Wnt protein.
作者信息
Hsieh J C, Rattner A, Smallwood P M, Nathans J
机构信息
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
出版信息
Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3546-51. doi: 10.1073/pnas.96.7.3546.
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 approximately 9 nM.
由于难以获得大量可溶的、具有生物活性的Wnt蛋白，Wnt信号通路的生化研究受到了阻碍。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）。表位标记或碱性磷酸酶标记的XWnt8蛋白由浓缩的S2细胞分泌，其形式适合进行定量生化实验，产量分别为每升5毫克和0.5毫克。还描述了在293细胞中产生小鼠卷曲蛋白8富含半胱氨酸结构域（CRD）的IgG融合蛋白的条件，产量为20毫克/升。我们展示了使用这些蛋白来研究可溶性XWnt8与各种卷曲蛋白、膜锚定或分泌的CRD以及果蝇卷曲蛋白2的CRD中的一组插入突变体之间的相互作用。在固相结合试验中，XWnt8-碱性磷酸酶融合蛋白对纯化的小鼠卷曲蛋白8-CRD-IgG融合蛋白的亲和力约为9 nM。
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