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.
Highlights
Protein-protein interactions (PPIs) play important roles in many cellular processes
bimolecular fluorescence complementation (BiFC) assay [6], it has been used in the investigation of subcellular localization of PPIs and their regulation mechanisms in living cells, especially the PPIs occurring on the cell membrane or with weak affinity [7,8,9,10]
The high contrast in BiFC efficiency between the positive and negative control was mainly due to the decreased BiFC signal in the negative control (ΔFos/Jun), where less than 2% cells had detectable BiFC signal
Summary
Protein-protein interactions (PPIs) play important roles in many cellular processes. To visualise the mechanisms and function roles of PPIs directly, various methods such as bimolecular fluorescence complementation (BiFC) [1,2], and fluorescence resonance energy transfer (FRET) [3], have been developed. (1) using a lower concentration of plasmids to reduce the expression of the fusion proteins and decrease the chance of spontaneous association of FP fragments [8]; (2) using mutation technology by replacing some critical amino acid of the split fluoresent protein to reduce self-assembly can increase signal to noise ratios in the Venus-based BiFC system [15,16]. While non-specific FP fragment associations can be mitigated by ensuring that the proteins of interest are expressed at lower concentrations, the false positive risks of BiFC assay still exist, which may confound identification of unknown or weak PPIs. In recent years, many modifications and enhancements to BiFC assay have been developed [1,2,17].
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