Abstract
BackgroundMost of the biological processes rely on the formation of protein complexes. Investigation of protein-protein interactions (PPI) is therefore essential for understanding of cellular functions. It is advantageous to perform mammalian PPI analysis in mammalian cells because the expressed proteins can then be subjected to essential post-translational modifications. Until now mammalian two-hybrid assays have been performed on individual gene scale. We here describe a new and cost-effective method for the high-throughput detection of protein-protein interactions in mammalian cells that combines the advantages of mammalian two-hybrid systems with those of DNA microarrays.ResultsIn this cell array protein-protein interaction assay (CAPPIA), mixtures of bait and prey expression plasmids together with an auto-fluorescent reporter are immobilized on glass slides in defined array formats. Adherent cells that grow on top of the micro-array will become fluorescent only if the expressed proteins interact and subsequently trans-activate the reporter. Using known interaction partners and by screening 160 different combinations of prey and bait proteins associated with the human androgen receptor we demonstrate that this assay allows the quantitative detection of specific protein interactions in different types of mammalian cells and under the influence of different compounds. Moreover, different strategies in respect to bait-prey combinations are presented.ConclusionWe demonstrate that the CAPPIA assay allows the quantitative detection of specific protein interactions in different types of mammalian cells and under the influence of different compounds. The high number of preys that can be tested per slide together with the flexibility to interrogate any bait of interest and the small amounts of reagents that are required makes this assay currently one of the most economical high-throughput detection assays for protein-protein interactions in mammalian cells.
Highlights
Most of the biological processes rely on the formation of protein complexes
Using known interaction partners and by screening 160 different combinations of prey and bait proteins associated with the human androgen receptor we demonstrate that this assay allows the quantitative detection of specific protein interactions in different types of mammalian cells and under the influence of different compounds
We demonstrate that the cell array protein-protein interaction assay (CAPPIA) assay allows the quantitative detection of specific protein interactions in different types of mammalian cells and under the influence of different compounds
Summary
Most of the biological processes rely on the formation of protein complexes. Investigation of protein-protein interactions (PPI) is essential for understanding of cellular functions. In classical yeast or mammalian two-hybrid based assays typically two proteins of interest are ectopically expressed as fusion proteins, one with the DNA Binding Domain (DBD) of for example GAL4 or LexA and the other with a transcriptional Activating Domain (AD), such that if both proteins show any interaction, the DBD and AD are functionally linked together at the promoter, reconstituting transcriptional activity [1,2,3]. This causes reporters that contain GAL4- or LexA binding sequences to be transcribed. Until now high-throughput analyses of mammalian protein interactions were typically performed in yeast [5,6] and putative interactions were confirmed in mammalian two-hybrid assays on a gene-by-gene scale [7,8]
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