High-throughput mammalian two-hybrid screening for protein-protein interactions using transfected cell arrays.
BACKGROUND
Most 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.


RESULTS
In 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.


CONCLUSIONS
We 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.
Fiebitz
Andrea
A
Max Planck Institute for Molecular Genetics, Department Vertebrate Genomics, Fabeckstr, 60-62, 14195 Berlin, Germany. fiebitz@molgen.mpg.de
Nyarsik
Lajos
L
Haendler
Bernard
B
Hu
Yu-Hui
YH
Wagner
Florian
F
Thamm
Sabine
S
Lehrach
Hans
H
Janitz
Michal
M
Vanhecke
Dominique
D
D000728:Androgens / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D020411:Oligonucleotide Array Sequence Analysis; D025941:Protein Interaction Mapping / Q000379:methods*; D011944:Receptors, Androgen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D020798:Two-Hybrid System Techniques
D000728:Androgens; D008024:Ligands; D011944:Receptors, Androgen; D011994:Recombinant Proteins