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,†email, 1,2,†email, 1email and 1,* email
Received: 4 December 2013; in revised form: 15 January 2014 / Accepted: 28 January 2014 / Published: 19 February 2014
(This article belongs to the Section Biosensors)
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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
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MDPI and ACS Style

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.

AMA Style

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(2):3284-3292.

Chicago/Turabian Style

Liu, Shun; Li, Xiangyong; Yang, Jie; Zhang, Zhihong. 2014. "Low False-Positives in an mLumin-Based Bimolecular Fluorescence Complementation System with a Bicistronic Expression Vector." Sensors 14, no. 2: 3284-3292.

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