Practical applications of green fluorescent protein ('GFP')-like fluorescent proteins (FPs) from species of the class Anthozoa (sea anemones, corals and sea pens) are strongly restricted owing to their oligomeric nature. Here we suggest a strategy to overcome this problem by the use of two covalently linked identical red FPs as non-oligomerizing fusion tags. We have applied this approach to the dimeric far-red fluorescent protein HcRed1 and have demonstrated superiority of the tandem tag in the in vivo labelling of fine cytoskeletal structures and tiny nucleoli. In addition, a possibility of effective fluorescence resonance energy transfer ('FRET') between enhanced yellow FP mutant ('EYFP') and tandem HcRed1 was demonstrated in a protease assay.
Abbreviations used: EGFP, enhanced green fluorescent protein; EYFP, enhanced yellow fluorescent protein; FP, fluorescent protein; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; LP, long-pass filter; ROI, region of interest; t-HcRed1, tandem HcRed1.