公开(公告)号：EP2087355A2

公开(公告)日：2009-08-12

申请号：EP07859356.3

申请日：2007-11-27

申请人： CIS bio international ,  Trinquet, Eric ,  Brinker, Achim ,  Claret, Emmanuel ,  Mathis, Gérard

发明人： TRINQUET, Eric ,  BRINKER, Achim ,  CLARET, Emmanuel ,  MATHIS, Gérard

IPC分类号： G01N33/574

CPC分类号： G01N33/542 ,  C12Q1/37 ,  C12Q1/48 ,  C12Q1/485

摘要： Method for the detection in homogeneous medium of a post-translational modification of a protein substrate catalyzed by a cell enzyme, characterized in that the post-translational modification reaction takes place in intact living cells, in that these cells comprise a heterologous expression vector coding for a fusion protein comprising the protein substrate and a first coupling domain and in that it comprises the following stages: (v) Incubation of the cells in the presence or in the absence of a compound to be tested capable of modulating the activity of said enzyme, (vi) Addition to the reaction medium of a first fluorescent compound member of a first pair of FRET partners covalently bonded to a coupling agent capable of binding specifically to the first coupling domain present on the protein substrate, (vii) Addition to the reaction medium of a second fluorescent compound member of this first pair of FRET partners, and covalently bonded to a binding domain specific to the site of the protein substrate having undergone the post-translational modification and not binding to the non-modified protein substrate, (i) Measurement of the FRET signal emitted by the sample, this signal being representative of the quantity of protein substrate having undergone said post-translational modification; and cells for the implementation of said method.