A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells.
Green fluorescent protein (GFP) and GFP-like proteins of different colors are important tools in cell biology. In many studies, the intracellular targeting of proteins has been determined by transiently expressing GFP fusion proteins and analyzing their intracellular localization by fluorescence microscopy. In most vectors, expression of GFP is driven by the enhancer/promoter cassette of the immediate early gene of human cytomegalovirus (hCMV). This cassette generates high levels of protein expression in most mammalian cell lines. Unfortunately, these nonphysiologically high protein levels have been repeatedly reported to artificially alter the intracellular targeting of proteins fused to GFP. To cope with this problem, we generated a multitude of attenuated GFP expression vectors by modifying the hCMV enhancer/promoter cassette. These modified vectors were transiently expressed, and the expression levels of enhanced green fluorescent protein (EGFP) alone and enhanced yellow fluorescent protein (EYFP) fused to another protein were determined by fluorescence microscopy and/or Western blotting. As shown in this study, we were able to (i) clearly reduce the expression of EGFP alone and (ii) reduce expression of an EYFP fusion protein down to the level of the endogenous protein, both in a graded manner.
Institute of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany. nalaskowski@uke.uni-hamburg.de
Marcus M
MM
Nalaskowski
Patrick
P
Ehm
Susanne
S
Giehler
Georg W
GW
Mayr
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D015153:Blotting, Western; D002460:Cell Line; D002477:Cells / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008322:Mammals / Q000378:metabolism*; D016296:Mutagenesis / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism*; D017384:Sequence Deletion / Q000235:genetics
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins