Quantitative Analyse von Proteininteraktionen

Coordinator:    Dr. Jörg Hoheisel
Institution: Deutsches Krebsforschungszentrum
The major objective of this subproject is the quantitative determination of protein interactions with other proteins and DNA on a genome-wide scale, especially in view of the effects of treatments with particular compounds or drugs. On the basis of an established process for the production of proteins by an in situ transcription and translation technique, we will establish comprehensive protein microarrays from full-length cDNA libraries, covering many genes/proteins of the human genome. On the basis of these comprehensive protein microarrays, we will analyse protein-protein interactions. As part of this, we will perform dissociation analyses, determining binding constants from the observed dissociation behaviour. One application will be the analysis of the affinity and specificity of antibodies produced for imaging or other purposes. Also, the interaction of regulatory proteins will be investigated, with an emphasis on human transcription factors. For studying the effects of patient-specific protein sequence modifications in individual tissues, we will combine the in situ production of protein microarrays with the in situ production on the microarray of PCR-products. Fitting primer pairs are spotted or synthesised in one spot of an array. Upon hybridisation of RNA or cDNA, PCR is performed on the chip. Performing such a reaction with RNA/cDNA from individual tissues, the relevant protein can subsequently be produced by in situ transcription/translation. Thereby protein microarrays representing gene products of individual tissues or patients can be produced and utilised for an analysis of individual proteomes. The approach will also be used for studies on the mouse models. Taking advantage of a small-molecule library of currently 55,000 compounds that is available at DKFZ, the effect of particular basic compound structures or – alternatively – particular drugs on the interaction of proteins will be performed. One area of emphasis will be the interactions of transcription factors, given their special position in the hierarchy of regulative processes with binding to both DNA and other proteins.
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