Methods for cell screening of compounds capable of...

C - Chemistry – Metallurgy – 12 – N

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C12N 15/52 (2006.01) A61P 1/00 (2006.01) C07K 14/395 (2006.01) C12N 15/62 (2006.01) C12N 15/63 (2006.01) C12N 15/81 (2006.01) C12Q 1/02 (2006.01) C12Q 1/68 (2006.01)

Patent

CA 2393115

The invention concerns methods for cell screening of agents capable of modulating the activity of SCF?Met30¿ complexes comprising the following steps: (i) contacting the product to be tested with a modified yeast strain, including (a) a hybrid sequence comprising a sequence coding for a Met4 protein, in its wild or mutated form, fused in phase with at least a sequence coding for an appropriate marker, said hybrid sequence being expressed under the control of a promoter, active in the yeast and optionally (b) a reporter transcriptional system, consisting of a reporter gene placed under the control of an appropriate operating sequence or an appropriate yeast promoter, (ii) adding methionine and (iii) determining the level of expression and stability of the expressed protein from the hybrid sequence, and their uses. The invention also concerns plasmids and yeast strains capable of being used in said methods.

Procédés de criblage cellulaire d'agents aptes à moduler l'activité des complexes SCFMet30, incluant les étapes suivantes : (i) la mise en contact du produit à tester avec une souche de levure modifiée, comportant (a) une séquence hybride, comprenant une séquence codant pour une protéine Met4, sous sa forme sauvage ou mutée, fusionnée en phase avec au moins une séquence codant pour un marqueur approprié, ladite séquence hybride étant exprimée sous le contrôle d'un promoteur, actif chez la levure et éventuellement (b) un système transcriptionnel rapporteur, constitué par un gène rapporteur placé sous le contrôle d'une séquence opératrice appropriée ou d'un promoteur de levure approprié, (ii) l'addition de méthionine et (iii) la détermination du niveau d'expression et de la stabilité de la protéine exprimée à partir de la séquence hybride, et leurs utilisations. Plasmides et souches de levures aptes à être utilisés dans ces procédés.

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