| 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.
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