Identification of protein-protein interactions in live mammalian cells offers significant advantages over existing biochemical (eg affinity purification/mass spec) and genetic (e.g. yeast 2 hybrid) assays by preserving cellular context and protein localisation. BiFC enables direct visualization of protein interactions in living cells through the folding of two non-fluorescent fragments to produce a functional fluorescent protein when brought into proximity by an in vivo interaction between the two proteins fused to the fragments. We have extended the BiFC principle by using the human ORFeome, a collection of ~15,000 individually cloned and annotated cDNAs, to generate a BiFC fusion library for interaction screening by high-throughput recombination cloning. The BiFC-ORF library will provide a resource for rapid investigation of binary protein interactions (ie with candidate bait/prey proteins) in living cells, as well as large-scale screens for interacting proteins. The system is easily adaptable to numerous applications by simply using different “bait” proteins, which can also be rapidly generated using recombination cloning.
Last updated on March 13, 2013 @9:07 am