A Novel Pair of Split Venus Fragments to Detect Protein–Protein Interactions by In Vitro and In Vivo Bimolecular Fluorescence Complementation Assays
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Protein–protein interactions are critical components of almost every cellular process. The bimolecular fluorescence complementation (BiFC) method has been used to detect protein–protein interactions in both living cells and cell-free systems. The BiFC method is based on the principle that a fluorescent protein is reassembled from its two complementary non-fluorescent fragments when an interaction occurs between two proteins, each one fused to each fragment. In vivo and in vitro BiFC assays, which use a new pair of split Venus fragments composed of VN210 (amino acids 1–210) and VC210 (amino acids 210–238), are useful tools to detect and quantify various protein–protein interactions (including the cofilin–actin and Ras–Raf interactions) with high specificity and low background fluorescence. Moreover, these assays can be applied to screen small-molecule inhibitors of protein–protein interactions.
Key wordsBimolecular fluorescence complementation (BiFC) Split Venus Protein–protein interaction High-throughput screening Cofilin Actin
We are grateful to Atsushi Miyawaki for providing Venus cDNAs, Takao Yamori for supplying the SCADS inhibitor kits, and Masato Oikawa for providing a library of chemical compounds. We also thank Tai Kiuchi, Kazuyasu Shoji, and Kaori Sampei for suggestions. This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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