The Renilla luciferase-modified GFP fusion protein is functional in transformed cells

The cDNA of Renilla reniformis luciferase (ruc) has been cloned and used successfully as a marker gene in a variety of transgenic species. Similarly, the transfer and expression of green fluorescent protein (GFP) cDNA (gfp) and its mutants from Aeguorea victoria resulted in high levels of GFP in transformed cells, allowing convenient visualization of gene expression under the microscope. Here we present the construction of four fusion genes from the cDNAs of Renilla luciferase and Aequorea GFP mutants (gfp2 and gfph, which have been engineered specifically for expression in prokaryotic organisms and mammalian cells, respectively). The fusion gene I (rg2) contains the Renilla luciferase cDNA linked through a 15-nucleotide (5 amino acid) spacer added to its 3' end to the 5' end of the intact gfp2. When the gfph fragment replaces gfp2, fusion gene II (rg) was formed. In fusion gene III (g2r), the positions of the ruc and gfp2 are reversed with a linker composed of seven amino acids in length. In fusion gene IV (gr), gfph replaced gfp2 with a linker of nine amino acids. The fusion gene cassettes I and III were placed into pBluescript KS II (+) and the fusion gene II and IV into mammalian expression vector pCEP4. The above plasmids were transformed into E. coli, different mammalian cell lines, and mouse embryos (microinjection). Fusion proteins with an apparent molecular weight around 65 kDa were detected by Western blotting using either anti-Renilla luciferase antibody or anti-Aequorea GFP antibody. Proteins RG2 and G2R extracted from the transformed E. coli have both green fluorescence activity and luciferase activity when expressed in E. coli. RG and GR are active in mammalian cells, ES cells, and mouse embryos. Fluorescence resonance energy transfer (FRET) between Renilla luciferase (emission at 478 nm) and Aequorea GFP (emission 510 nm) was detected by spectrofluorimetry, only if the two proteins were linked. The Renilla luciferase-GFP fusion proteins offer a novel marker system for photosynthetic microorganisms and plants. This fusion protein helps to overcome the problems in quantifying GFP fluorescence. The determination effusion protein in cells can be quantified based on luciferase activity. Furthermore, the system may be useful in the study of protein-protein interactions in vivo.