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    METHODS AND COMPOSITIONS RELATED TO IDENTIFYING PROTEIN-PROTEIN INTERACTIONS
    8.
    发明申请
    METHODS AND COMPOSITIONS RELATED TO IDENTIFYING PROTEIN-PROTEIN INTERACTIONS 审中-公开
    鉴定蛋白质 - 蛋白质相互作用相关的方法和组合

    公开(公告)号：US20090221440A1

    公开(公告)日：2009-09-03

    申请号：US11572088

    申请日：2005-07-12

    申请人： Zhiyong Ding ,  Gordon Mills

    发明人： Zhiyong Ding ,  Gordon Mills

    IPC分类号： C40B30/06 ,  C40B40/02 ,  C12N15/86 ,  C12N15/85 ,  C12N5/10

    CPC分类号： C12N15/1055

    摘要： The present invention involves compositions and methods for assessing protein interactions in eukaryotic cells. Certain embodiments involve Retrovirus-Based Molecular Two-Hybrid Screens (ReMTH). ReMTH screens differ from other methods by performing screens in the native cellular hosts without cDNA library construction. Embodiments of the invention use the advantages of tagging endogenous genes with an exon encoding a marker fragment in combination with protein-fragment complementation assays (PCAs), which includes the complementation of at least two marker fragments to form a detectable marker complex. ReMTH vectors insert a nucleotide sequence encoding a first fragment of a marker, such as green fluorescent protein (GFP), into an endogenous gene resulting in expression of random endogenous genes tagged with a first marker fragment forming an endogenous prey protein or prey protein. ReMTH contain cells also express a bait protein that is fused with a second marker fragment. Prey/Bait interaction produces a reconstituted, detectable marker.

    摘要翻译： 本发明涉及用于评估真核细胞中蛋白质相互作用的组合物和方法。 某些实施方案涉及基于逆转录病毒的分子双杂交筛选（ReMTH）。 ReMTH屏幕与其他方法不同，通过在没有cDNA文库构建的天然细胞宿主中进行筛选。 本发明的实施方案利用将编码标记片段的外显子与蛋白质 - 片段互补测定（PCAs）结合的内源基因标记的优点，其包括至少两个标记片段的互补以形成可检测的标记物复合物。 ReMTH载体将编码标记物的第一片段（例如绿色荧光蛋白（GFP））的核苷酸序列插入到内源基因中，导致用形成内源猎物蛋白质或猎物蛋白质的第一标记片段标记的随机内源基因的表达。 含有细胞的ReMTH还表达与第二标记片段融合的诱饵蛋白质。 捕食/诱饵相互作用产生重建的可检测标记。