公开(公告)号：EP1677113A1

公开(公告)日：2006-07-05

申请号：EP04030994.0

申请日：2004-12-29

申请人： Max-Delbrück-Centrum für Molekulare Medizin (MDC)

发明人： Wanker, Erich ,  Lalowski, Maciej ,  Stelzl, Ulrich ,  Strödicke, Martin ,  Hänig, Christian ,  Worm, Uwe ,  Göhler, Heike

IPC分类号： G01N33/68 ,  C12N15/10 ,  C12N15/00 ,  C12N5/00 ,  C07K16/00 ,  C07K14/00 ,  A61K38/00 ,  A61K39/395 ,  A61K48/00

CPC分类号： C12N15/1055

摘要： The present invention relates to a method for identification of a protein-protein interaction of protein interaction partners in a disease-related network of proteins comprising the steps of (a) identifying nucleic acid molecules by at least partial 5'sequencing and, optionally, additionally adding recombinantly cloned and sequenced nucleic acid molecules, wherein said nucleic acid molecules encode a selection of proteins suspected to contain one or several of said interaction partners and wherein said nucleic acid molecules are annotated with a positional information; (b) in frame cloning of the nucleic acid molecules of step (a) into prey and bait vectors, wherein one copy of each nucleic acid molecule is cloned into said prey vector and a second copy of each nucleic acid molecule is cloned into said bait vector; (c) transforming a first suitable host cell with the prey vector obtained in step (b) and a second suitable host cell with the bait vector obtained in step (b), wherein said first and said second host cell have a different genetic constitution and can be mated; (d) mating the first suitable host cell of step (c) with the second suitable host cell of step (c), and expressing the proteins encoded by the nucleic acid molecules obtained in step (b); (e) selecting the mated host cell obtained in step (d) on the basis of a selection advantage which is caused by the protein-protein interaction between the protein interaction partner encoded by the nucleic acid molecule of the prey vector contained in said cell and the protein interaction partner encoded by the nucleic acid molecule of the bait vector contained in said cell; and (f) identifying with the positional information obtained in step (a) the protein interaction partners of step (e) and thereby identifying the protein-protein interaction.

摘要翻译： 本发明涉及用于鉴定蛋白质相互作用伴侣蛋白质相互作用的蛋白质相互作用的方法，所述方法包括以下步骤：（a）通过至少部分的5'-测序鉴定核酸分子，以及任选地另外 加入重组克隆和测序的核酸分子，其中所述核酸分子编码怀疑含有一个或几个所述相互作用配偶体的蛋白质的选择，并且其中所述核酸分子用位置信息注释; （b）将步骤（a）的核酸分子的框架克隆入猎物和诱饵载体，其中每个核酸分子的一个拷贝被克隆到所述猎物载体中，并且将每个核酸分子的第二个拷贝克隆到所述诱饵中 向量; （c）用步骤（b）中获得的猎物载体转化第一合适的宿主细胞，和用步骤（b）中获得的诱饵载体的第二合适的宿主细胞转化，其中所述第一和所述第二宿主细胞具有不同的遗传构成， 可以交配; （d）将步骤（c）的第一合适宿主细胞与步骤（c）的第二适合宿主细胞交配，并表达由步骤（b）中获得的核酸分子编码的蛋白质; （e）基于由所述细胞中包含的被捕获载体的核酸分子编码的蛋白质相互作用伴侣之间的蛋白质 - 蛋白质相互作用引起的选择优势，选择步骤（d）中获得的配对宿主细胞;以及 由所述细胞中所含的诱饵载体的核酸分子编码的蛋白质相互作用伴侣; 和（f）用步骤（a）中获得的位置信息鉴定步骤（e）的蛋白质相互作用伴侣，从而鉴定蛋白质 - 蛋白质相互作用。