An improved mRFP1 adds red to bimolecular fluorescence complementation

Abstract

Protein-protein interactions are fundamental to virtually every aspect of cellular functions. Blue, green and yellow bimolecular fluorescence complementation (BiFC) systems based on GFP and its variants allow the investigation of protein-protein interactions in vivo. We have developed the first red BiFC system based on an improved monomeric red fluorescent protein (mRFP1-Q66T), expanding the range of possible applications for BiFC.With interactome data available for several model organisms, a challenging next step in post-genomic research is to analyze protein complex formation in vivo. The recently developed BiFC assay is a comparably fast and simple noninvasive technology to study protein interactions inside living cells. BiFC is based on the reconstitution of the yellow fluorescent protein (YFP) from two nonfluorescent fragments when they are brought into close proximity by a physical interaction between proteins fused to each fragment 1 . This approach has proven to be robust and versatile, and multicolor versions using spectral variants of GFP further increase the potential of this technology 2 . The color spectrum available for BiFC, however, has been limited to blue, green and yellow.The red fluorescent protein from Discosoma sp. (DsRED) and its variants are established intracellular reporter proteins, but the characteristics of most DsRED variants, particularly their obligate tetramerization, impede their application in BiFC 3,4 . An extensively mutated monomeric DsRED variant (mRFP1) has been generated 5 , but substantially altered spectra, poor brightness and low photostability limit its usefulness as a reporter protein.We have identified improved mRFP1 mutants, which allowed us to establish a red fluorescent reporter system for detection of protein interactions in vivo. We used site-directed mutagenesis to generate mRFP1 variants with a randomized first position of the fluorophore. Screening of 5,000 colonies resulted in the identification of 50 clones exhibiting strong red fluorescence representing three different mutations (Q66C, Q66S and Q66T) with frequencies of 5%, 43% and 52%, respectively.

Gateway Vectors for Plant Genetic Engineering: Overview of Plant Vectors, Application for Bimolecular Fluorescence Complementation (BiFC) and Multigene Construction

“…However, other fluorescent proteins, BFP (Hu & Kerppola, 2003), CFP (Kodama & Wada, 2009;Lee et al, 2008), GFP (Hu et al, 2002;Kodama & Wada, 2009), Venus, (Lee et al, 2008), Cerulean (Lee et al, 2008), DsRed-monomer (Kodama & Wada, 2009), mRFP1 (Jach et al, 2006), mCherry (Fan et al, 2008), and a far-red fluorescent protein, mLumin (Chu et al, 2009), have reportedly been useful for BiFC assay. We adopted CFP, GFP, YFP and mRFP1 to generate vectors ( Figure 9B), and verified their usefulness for detection of protein-protein interactions ( Figure 10B-E).…”

Improvement of a Venus-Based Bimolecular Fluorescence Complementation Assay to Visualize bFos-bJun Interaction in Living Cells

“…[2][3][4][5][6][7][8][9] Venus, a bright-yellow variant of GFP, efficiently matures at physiological temperature, and the intensity of fluorescence is higher than EYFP. 10) Thus Venus is suitable for the visualization of cellular structures.…”

A bright green-colored bimolecular fluorescence complementation assay in living plant cells

“…To date, over 10 fluorescent proteins have been used for the BiFC assay . For example, cyan (Hu and Kerppola 2003;Shyu et al 2006), green (Ghosh et al 2000), red (Chu et al 2009;Fan et al 2008;Jach et al 2006;Kodama and Wada 2009) and photoswitchable (Lee et al 2010) fluorescent protein-based BiFC assays are available. Using these fluorescence complementation technologies, advanced BiFC-based techniques such as multicolor BiFC and BiFC-based fluorescence (Förster) resonance energy transfer (FRET) assays have also been developed.…”