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Sensors 2014, 14(2), 3284-3292; doi:10.3390/s140203284
Communication

Low False-Positives in an mLumin-Based Bimolecular Fluorescence Complementation System with a Bicistronic Expression Vector

1,†
,
1,2,†
,
1
 and
1,*
1 Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China 2 Department of Biology, Dezhou University, Dezhou 253023, China These authors contributed equally to this work.
* Author to whom correspondence should be addressed.
Received: 4 December 2013 / Revised: 15 January 2014 / Accepted: 28 January 2014 / Published: 19 February 2014
(This article belongs to the Section Biosensors)
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Abstract

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 (ΔFos/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.
Keywords: protein-protein interaction; bimolecular fluorescence complementation (BiFC); expression vector; false positive; fluorescent protein protein-protein interaction; bimolecular fluorescence complementation (BiFC); expression vector; false positive; fluorescent protein
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

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Liu, S.; Li, X.; Yang, J.; Zhang, Z. Low False-Positives in an mLumin-Based Bimolecular Fluorescence Complementation System with a Bicistronic Expression Vector. Sensors 2014, 14, 3284-3292.

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