SOLUBLE, HIGHLY FLUORESCENT VARIANTS OF GREEN FLUORESCENT PROTEIN (GFP) FOR USE IN HIGHER-PLANTS

Green fluorescent protein (GFP) from Aequorea victoria has rapidly bec ome a standard reporter in many biological systems. However, the use o f GFP in higher plants has been limited by aberrant splicing of the co rresponding mRNA and by protein insolubility. It has been shown that G FP can be expressed in Arabidopsis thaliana after altering the codon u sage in the region that is incorrectly spliced, but the fluorescence s ignal is weak, possibly due to aggregation of the encoded protein. Thr ough site-directed mutagenesis, we have generated a more soluble versi on of the codon-modified GFP called soluble-modified GFP (smGFP). The excitation and emission spectra for this protein are nearly identical to wild-type GFP. When introduced into A. thaliana, greater fluorescen ce was observed compared to the codon-modified GFP, implying that smGF P is 'brighter' because more of it is present in a soluble and functio nal form. Using the smGFP template, two spectral variants were created , a soluble-modified red-shifted GFP (smRS-GFP) and a soluble-modified blue-fluorescent protein (smBFP). The increased fluorescence output o f smGFP will further the use of this reporter in higher plants. In add ition, the distinct spectral characters of smRS-GFP and smBFP should a llow for dual monitoring of gene expression, protein localization, and detection of in vivo protein-protein interactions.