{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,11]],"date-time":"2026-06-11T14:07:09Z","timestamp":1781186829933,"version":"3.54.1"},"reference-count":24,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2014,2,19]],"date-time":"2014-02-19T00:00:00Z","timestamp":1392768000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/3.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Sensors"],"abstract":"<jats:p>The simplicity and sensitivity of the bimolecular fluorescence complementation (BiFC) assay make it a powerful tool to investigate protein-protein interactions (PPIs) in living cells. However, non-specific association of the fluorescent protein fragments in a BiFC system can complicate evaluation of PPIs. Here, we introduced a bicistronic expression vector, pBudCE4.1, into an mLumin-based BiFC system, denoted as the  BEVL-BiFC system. The BEVL-BiFC system achieved a 25-fold contrast in BiFC efficiency between positive (Fos\/Jun) and negative (\u0394Fos\/Jun) PPIs. The high BiFC efficiency was due to a low false-positive rate, where less than 2% of cells displayed BiFC in the negative control. K-Ras and its interactive proteins, Ras binding domain (RBD) of Raf-1 and Grb2 were used to confirm the accuracy of the BEVL-BiFC system. The results also provide direct evidence in individual cells that post-translational modification of  K-Ras and its localization at the plasma membrane (PM) were not essential for the interaction of K-Ras and Raf-1, whereas the interaction of Grb2 and K-Ras did depend on the PM localization of K-Ras. Taken together, the BEVL-BiFC system was developed to reduce the false-positive phenomenon in BiFC assays, resulting in more robust and accurate measurement of PPIs in living cells.<\/jats:p>","DOI":"10.3390\/s140203284","type":"journal-article","created":{"date-parts":[[2014,2,19]],"date-time":"2014-02-19T11:10:21Z","timestamp":1392808221000},"page":"3284-3292","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Low False-Positives in an mLumin-Based Bimolecular Fluorescence Complementation System with a Bicistronic Expression Vector"],"prefix":"10.3390","volume":"14","author":[{"given":"Shun","family":"Liu","sequence":"first","affiliation":[{"name":"Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for  Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiangyong","family":"Li","sequence":"additional","affiliation":[{"name":"Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for  Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China"},{"name":"Department of Biology, Dezhou University, Dezhou 253023, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jie","family":"Yang","sequence":"additional","affiliation":[{"name":"Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for  Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhihong","family":"Zhang","sequence":"additional","affiliation":[{"name":"Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for  Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2014,2,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2876","DOI":"10.1039\/b909638h","article-title":"Visualization of molecular interactions using bimolecular fluorescence complementation analysis: Characteristics of protein fragment complementation","volume":"38","author":"Kerppola","year":"2009","journal-title":"Chem. Soc. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"285","DOI":"10.2144\/000113943","article-title":"Bimolecular fluorescence complementation (bifc): A 5-year update and future perspectives","volume":"53","author":"Kodama","year":"2012","journal-title":"Biotechniques"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Wang, X.P., Yu, H.N., and Chen, T.S. (2012). Quantitative fret measurement based on confocal microscopy imaging and partial acceptor photobleaching. J. Innov. Opt. Heal. Sci.","DOI":"10.1142\/S1793545812500150"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"45","DOI":"10.2144\/000113777","article-title":"Visualization of cofilin-actin and ras-raf interactions by bimolecular fluorescence complementation assays using a new pair of split venus fragments","volume":"52","author":"Ohashi","year":"2012","journal-title":"Biotechniques"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1078","DOI":"10.1016\/j.chembiol.2013.06.009","article-title":"A near-infrared bifc reporter for in vivo imaging of protein-protein interactions","volume":"20","author":"Filonov","year":"2013","journal-title":"Chem. Biol."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Kerppola, T. (2006). Complementary methods for studies of protein interactions in living cells. Nat. Method.","DOI":"10.1038\/nmeth1206-969"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Hemerka, J.N., Wang, D., Weng, Y., Lu, W., Kaushik, R.S., Jin, J., Harmon, A.F., and Li, F. (2009). Detection and characterization of influenza a virus pa-pb2 interaction through a bimolecular flouresence complementation assay. J. Virol.","DOI":"10.1128\/JVI.02300-08"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"449","DOI":"10.1038\/nrm1929","article-title":"Visualization of molecular interactions by fluorescence complementation","volume":"7","author":"Kerppola","year":"2006","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1016\/j.cell.2004.09.012","article-title":"Combinatorial marking of cells and organelles with reconstituted fluorescent proteins","volume":"119","author":"Zhang","year":"2004","journal-title":"Cell"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"738","DOI":"10.1111\/j.1476-5381.2009.00480.x","article-title":"Bimolecular fluorescence complementation: Lighting up seven transmembrane domain receptor signalling networks","volume":"159","author":"Rose","year":"2010","journal-title":"Br. J. Pharmacol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"61","DOI":"10.2144\/000112036","article-title":"Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions","volume":"40","author":"Shyu","year":"2006","journal-title":"Biotechniques"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"234","DOI":"10.1016\/j.bios.2009.06.008","article-title":"A novel far-red bimolecular fluorescence complementation system that allows for efficient visualization of protein interactions under physiological conditions","volume":"25","author":"Chu","year":"2009","journal-title":"Biosens. Bioelectron"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1278","DOI":"10.1038\/nprot.2006.201","article-title":"Design and implementation of bimolecular fluorescence complementation (bifc) assays for the visualization of protein interactions in living cells","volume":"1","author":"Kerppola","year":"2006","journal-title":"Nat. Protoc."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1038\/nbt1044","article-title":"Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein","volume":"23","author":"Cabantous","year":"2005","journal-title":"Nat. Biotechnol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"272","DOI":"10.3109\/10520295.2010.483068","article-title":"Lec-bifc: A new method for rapid assay of protein interaction","volume":"86","author":"Lin","year":"2011","journal-title":"Biotech. Histochem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"793","DOI":"10.2144\/000113519","article-title":"An improved bimolecular fluorescence complementation assay with a high signal-to-noise ratio","volume":"49","author":"Kodama","year":"2010","journal-title":"Biotechniques"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"465","DOI":"10.1146\/annurev.biophys.37.032807.125842","article-title":"Biomolecular fluorescence complementation (bifc) analysis as a probe of protein interactions in living cells","volume":"37","author":"Kerppola","year":"2008","journal-title":"Annu. Rev. Biophys."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Zheng, Y., Lin, Q.Y., Jin, H.L., Chen, J., and Zhang, Z.H. (2012). Visualization of head and neck cancer models with a triple fusion reporter gene. J. Innov. Opt. Heal Sci.","DOI":"10.1142\/S1793545812500289"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Zheng, Y., Huang, C., Cheng, Z.Y., and Chen, M. (2012). Establishment of visible animal metastasis models for human nasopharyngeal carcinoma based on a far-red fluorescent protein. J. Innov. Opt. Heal Sci.","DOI":"10.1142\/S1793545812500198"},{"key":"ref_20","unstructured":"Image J software. Available online: http:\/\/rsb.info.nih.gov\/ij."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1065","DOI":"10.1038\/35082594","article-title":"Spatio-temporal images of growth-factor-induced activation of ras and rap1","volume":"411","author":"Mochizuki","year":"2001","journal-title":"Nature"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1038\/ng1748","article-title":"Germline kras mutations cause noonan syndrome","volume":"38","author":"Schubbert","year":"2006","journal-title":"Nat. Genet."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/S0092-8674(00)80601-7","article-title":"New insights into the minireview interaction of ras with the plasma membrane","volume":"98","author":"Magee","year":"1999","journal-title":"Cell"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"381","DOI":"10.1016\/j.ymeth.2003.10.011","article-title":"A cdna library functional screening strategy based on fluorescent protein complementation assays to identify novel components of signaling pathways","volume":"32","author":"Remy","year":"2004","journal-title":"Methods"}],"container-title":["Sensors"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1424-8220\/14\/2\/3284\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T21:08:17Z","timestamp":1760216897000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1424-8220\/14\/2\/3284"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2014,2,19]]},"references-count":24,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2014,2]]}},"alternative-id":["s140203284"],"URL":"https:\/\/doi.org\/10.3390\/s140203284","relation":{},"ISSN":["1424-8220"],"issn-type":[{"value":"1424-8220","type":"electronic"}],"subject":[],"published":{"date-parts":[[2014,2,19]]}}}