Peptide-based method for monitoring gene expression in a host cell

摘要

The present invention relates a method for monitoring the expression level of a gene in a host cell by modulating the activity of a regulatory biomolecule, comprising the steps of: (a) transforming a cell expressing a regulatory biomolecule with a nucleic acid molecule comprising an open reading frame encoding an interaction partner of said biomolecule in expressible form, wherein (i) said regulatory biomolecule is either a nucleic acid binding molecule that effects its regulatory activity upon binding or an allosterically controlled ribonucleic acid molecule; and (ii) the interaction partner of the biomolecule is encoded by a nucleic acid molecule comprising: (1) a nucleic acid sequence encoding a tagged (poly)peptide, or (2) a nucleic acid sequence encoding a tagged (poly)peptide or a peptide tag, a selectable marker gene and additional nucleotide sequences for site specific, in-frame integration of said nucleic acid molecule into the coding sequence of at least one host (poly)peptide of interest, wherein said tag comprises the interacting residues of the interaction partner and (b) assessing the expression level of the gene. Furthermore, the present invention relates to a method of producing and/or selecting a compound capable of modulating the activity of a nucleic acid binding protein comprising the steps of: (a) conducting a selection of compounds with the nucleic acid binding target protein under conditions allowing an interaction of the compound and the nucleic acid binding protein; (b) removing unspecifically bound compounds; (c) detecting specific binding of compounds to the nucleic acid binding target protein; (d) expressing in a cell, the nucleic acid binding protein and providing in trans the coding sequence of at least one indicator gene, wherein said coding sequence is under control of the target sequence of the nucleic acid binding protein; (e) adding a candidate compound to the cell of step (d); (f) determining the amount or activity of the indicator protein, wherein a reduced or increased amount of indicator protein is indicative of compounds, capable of modulating the activity of the nucleic acid binding protein; (g) selecting compounds capable of modulating the activity of the nucleic acid binding protein. Moreover, the present invention relates to nucleic acid molecules, polypeptides, expression vectors, host cells, ensembles of host cells and a non-human animal comprising said host cells. Finally, the present invention relates to a kit comprising the nucleic acid molecule, the (poly)peptide, the vector, the host cell or the ensemble of host cells of the present invention in one or more containers.