A split enhanced green fluorescent protein-based reporter in yeast two-hybrid system.
We have developed a novel reporter system involving a yeast two-hybrid assay, which utilizes the reconstitution of the split EGFP reporter in order to characterize the relevant protein-protein interactions. To our knowledge, this study represents the first application of the split EGFP system as a read-out in a yeast two-hybrid assay. In comparison with the existing two-hybrid system, the bait and prey vectors were improved with regard to the reporter and the replication control element. As a result, the reconstituted EGFP has been observed to evidence a restored fluorescence upon protein-protein interactions in yeast, thereby allowing for the characterization of its interactor. The use of a split EGFP reporter has some salient advantages. Firstly, no substrates are required for the production of fluorescence. Secondly, low copy number plasmids may help to solve the protein toxicity problem, via the reduction of expression. Thirdly, this technique may prove useful in overcoming the autoactivation problem, due to the fact that the read-out of the yeast two-hybrid system is transcription-independent. Collectively, our results showed that the split EGFP reporter system might potentially be applied in yeast two-hybrid assays for the high-throughput screening of protein-protein interactions, with a simple and direct fluorescent read-out.
Park
Kyoungsook
K
BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
Yi
So Yeon
SY
Lee
Chang-Soo
CS
Kim
Kyoon Eon
KE
Pai
Hyun-Sook
HS
Seol
Dai-Wu
DW
Chung
Bong Hyun
BH
Kim
Moonil
M
D004926:Escherichia coli / Q000235:genetics; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D020798:Two-Hybrid System Techniques
D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins