A novel orange-colored bimolecular fluorescence complementation (BiFC) assay using monomeric Kusabira-Orange protein

Abstract

The bimolecular fluorescence complementation (BiFC) assay was developed as a tool for the visualization of protein–protein interactions in living cells. To date, many types of BiFC systems with distinct colors have been developed. Most of the colors in the visible spectrum have been used in BiFC assays, with the exception of orange. In this study, we developed an orange-colored BiFC system using the Kusabira-Orange (KO) protein from the stony coral Fungia concinna. To obtain bright BiFC fluorescence, we compared fluorescence intensities of two monomeric KO variants (mKO1 and mKO2) and identified mKO2 as brighter than mKO1. The optimal split site for mKO2-based BiFC was defined by a comparative analysis of complementation efficiency and a signal-to-noise ratio. The resulting mKO2-based BiFC system successfully demonstrated protein dimerization in plant cells as a model experiment. The novel mKO2-based BiFC system will expand the possibility of multicolor BiFC analysis.

Activation of the plant mevalonate pathway by extracellular ATP

“…This experiment was performed at the Molecular Cytology Core of the University of Missouri (https://research.missouri.edu/mcc/). Quantification of fluorescence intensity was performed with ImageJ software 79 .…”

The localization of phototropin to the plasma membrane defines a cold-sensing compartment in Marchantia polymorpha

“…Note that pMpGWB306 contains a resistance gene for the herbicide chlorsulfuron. For transient expression of mCherry in E. densa and M. polymorpha , pDONR207-mCherry ( 19 ) was recombined with the destination vector pGWT35S ( 42 ) via LR reaction to generate pGWT35S-mCherry.…”

Spatial proteomics of vesicular trafficking: coupling mass spectrometry and imaging approaches in membrane biology

“…Protein–protein interaction studies based on imaging rely on the visualization of proteins in their cellular states without tissue disruption or organelle isolation (Table 2 ). Classic techniques for detecting protein interactions employ protein‐fragment complementation assays, including bimolecular fluorescence complementation (BiFC) and the split luciferase system (also referred to as luciferase complementation imaging assays), which are the simplest and most popular methods to assess protein interactions in living plant cells (Fujii et al ., 2018 ). However, these methods suffer from low accuracy and are prone to false positive and false negative interactions.…”