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标题翻译： ZAP-70相互作用分子鉴定方法和ZAP-70纯化方法

公开(公告)号：WO2007137867A1

公开(公告)日：2007-12-06

申请号：PCT/EP2007/004880

申请日：2007-06-01

申请人： CELLZOME AG , KRUSE, Ulrich , RAMSDEN, Nigel , DREWES, Gerard , EBERHARD, Dirk

发明人： KRUSE, Ulrich , RAMSDEN, Nigel , DREWES, Gerard , EBERHARD, Dirk

IPC分类号： G01N33/574 , C12Q1/48 , C07K1/04 , G01N33/68

CPC分类号： C12Q1/485 , C12N9/1205 , G01N33/57426 , G01N2333/91215 , G01N2500/02 , G01N2500/10

摘要： The invention provides in a first aspect a method for the identification of an ZAP-70 interacting compound, comprising the steps of a) providing a protein preparation containing phosphorylated ZAP-70, b) contacting the protein preparation with aminopyrido-pyrimidine ligand 24 immobilized on a solid support under conditions allowing the formation of an aminopyrido-pyrimidine ligand 24 - phosphorylated ZAP-70 complex, c) incubating the aminopyrido-pyrimidine ligand 24 — phosphorylated ZAP-70 complex with a given compound, and d) determining whether the compound is able to separate phosphorylated ZAP-70 from the immobilized aminopyrido-pyrimidine ligand 24. In a second aspect, the present invention relates to A method for the identification of an ZAP-70 interacting compound, comprising the steps of a) providing a protein preparation containing phosphorylated ZAP-70, b) contacting the protein preparation with ligand aminopyrido-pyrimidine ligand 24 immobilized on a solid support and with a given compound under conditions allowing the formation of an aminopyrido-pyrimidine ligand 24 - phosphorylated ZAP-70 complex, and c) detecting the aminopyrido-pyrimidine ligand 24 - phosphorylated ZAP-70 complex formed in step b). hi a third aspect, the invention provides a method for the identification of an ZAP-70 interacting compound, comprising the steps of: a) providing two aliquots of a protein preparation containing phosphorylated ZAP-70, b) contacting one aliquot with aminopyrido-pyrimidine ligand 24 immobilized on a solid support under conditions allowing the formation of an aminopyrido-pyrimidine ligand 24 - phosphorylated ZAP-70 complex, c) contacting the other aliquot with aminopyrido-pyrimidine ligand 24 immobilized on a solid support and with a given compound under conditions allowing the formation of an aminopyrido-pyrimidine ligand 24 - phosphorylated ZAP-70 complex, and d) determining the amount of aminopyrido- pyrimidine ligand 24 - phosphorylated ZAP-70 complex formed in steps b) and c). In a forth aspect, the invention relates to a method for the identification of an ZAP-70 interacting compound, comprising the steps of: a) providing two aliquots comprising each at least one cell containing phosphorylated ZAP-70, b) incubating one aliquot with a given compound, c) harvesting the cells of each aliquot, d) lysing the cells in order to obtain protein preparations, e) contacting the protein preparations with aminopyrido-pyrimidine ligand 24 immobilized on a solid support under conditions allowing the formation of an aminopyrido-pyrimidine ligand 24 - phosphorylated ZAP-70 complex, and f) determining the amount of aminopyrido-pyrimidine ligand 24 - phosphorylated ZAP-70 complex formed in each aliquot in step e).

摘要翻译：本发明在第一方面提供了一种用于鉴定ZAP-70相互作用化合物的方法，其包括以下步骤：a）提供含有磷酸化ZAP-70的蛋白质制剂，b）使蛋白质制剂与固定在其上的氨基吡啶并 - 嘧啶配体24接触在允许形成氨基吡啶并嘧啶配体24-磷酸化ZAP-70复合物的条件下的固体支持物，c）将氨基吡啶并 - 嘧啶配体24-磷酸化ZAP-70复合物与给定化合物一起孵育，和d）测定化合物是否为能够从固定化的氨基吡啶并嘧啶配体24分离磷酸化的ZAP-70。在第二方面，本发明涉及鉴定ZAP-70相互作用化合物的方法，包括以下步骤：a）提供含有磷酸化ZAP-70，b）将蛋白质制备物与固定在固体支持物上的配体氨基吡啶并 - 嘧啶配体24接触，给定化合物在允许形成氨基吡啶并嘧啶配体24-磷酸化ZAP-70复合物的条件下，和c）检测步骤b）中形成的氨基吡啶并 - 嘧啶配体24-磷酸化ZAP-70复合物。第三方面，本发明提供了鉴定ZAP-70相互作用化合物的方法，包括以下步骤：a）提供含有磷酸化ZAP-70的蛋白质制剂的两个等分试样，b）使一个等分试样与氨基吡啶并 - 嘧啶配体24在允许形成氨基吡啶并 - 嘧啶配体24-磷酸化ZAP-70复合物的条件下固定在固体支持物上，c）将其它等分试样与固定在固相载体上的氨基吡啶并 - 嘧啶配体24和在给定化合物条件下接触允许形成氨基吡啶并 - 嘧啶配体24-磷酸化ZAP-70复合物，和d）测定步骤b）和c）中形成的氨基吡啶并 - 嘧啶配体24-磷酸化ZAP-70复合物的量。在第四方面，本发明涉及一种鉴定ZAP-70相互作用化合物的方法，其包括以下步骤：a）提供两个等分试样，其包含每个至少一个含有磷酸化ZAP-70的细胞，b）将一个等分试样与给定化合物，c）收获每个等分试样的细胞，d）裂解细胞以获得蛋白质制剂，e）使蛋白质制剂与固定在固体支持物上的氨基吡啶并 - 嘧啶配体24接触，条件允许形成氨基吡啶 - 嘧啶配体24-磷酸化ZAP-70复合物，和f）测定在步骤e）中在每个等分试样中形成的氨基吡啶并 - 嘧啶配体24-磷酸化ZAP-70复合物的量。

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