McDonald O B, Chen W J, Ellis B, Hoffman C, Overton L, Rink M, Smith A, Marshall C J, Wood E R
Division of Biochemistry, GlaxoWellcome Inc., Research Triangle Park, North Carolina, 27709-13398, USA.
Anal Biochem. 1999 Mar 15;268(2):318-29. doi: 10.1006/abio.1998.3030.
We have developed a quantitative scintillation proximity assay (SPA) that reproduces the Raf/MEK/ERK signal transduction pathway. The components of this assay include human cRaf1, MEK1, and ERK2 and a biotinylated peptide substrate for ERK2. cRaf1 was expressed as a his-tagged protein in insect cells in an active form. MEK1 and ERK2 were expressed in Escherichia coli as glutathione S-transferase (GST)-fusion proteins in their inactive forms. ERK2 was removed from the GST portion of the fusion protein by cleavage with thrombin protease. When the purified components are incubated together, cRaf-1 phosphorylates and activates MEK1, MEK1 phosphorylates and activates ERK2, and ERK2 phosphorylates the peptide, biotin-AAATGPLSPGPFA. Phosphorylation of the peptide using [gamma-33P]ATP is detected following binding to streptavidin-coated SPA beads. The assay detects inhibitors of cRaf1, MEK1, or ERK2, and has been used to screen large numbers of compounds. The specific target of inhibition was subsequently identified with secondary assays described herein.
我们开发了一种定量闪烁邻近分析（SPA）方法，该方法可重现Raf/MEK/ERK信号转导途径。此分析方法的组成部分包括人源cRaf1、MEK1和ERK2以及ERK2的生物素化肽底物。cRaf1在昆虫细胞中以带组氨酸标签的活性形式表达。MEK1和ERK2在大肠杆菌中以谷胱甘肽S-转移酶（GST）融合蛋白的非活性形式表达。通过凝血酶蛋白酶切割，将ERK2从融合蛋白的GST部分去除。当将纯化的组分一起孵育时，cRaf-1磷酸化并激活MEK1，MEK1磷酸化并激活ERK2，ERK2磷酸化肽生物素-AAATGPLSPGPFA。使用[γ-33P]ATP对肽进行磷酸化后，通过与链霉亲和素包被的SPA微球结合来检测。该分析方法可检测cRaf1、MEK1或ERK2的抑制剂，并已用于筛选大量化合物。随后通过本文所述的二级分析确定抑制的具体靶点。
