Green fluorescent protein: untapped potential in immunotechnology (original) (raw)
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Biotechnological applications of green fluorescent protein
Applied Microbiology and Biotechnology, 2003
Since its first use as a reporter gene in 1994, green fluorescent protein (GFP) has served as the researcher 0 s agent: slipping, virtually undetected, into unseen spaces, reporting back valuable information, and securing the delivery of precious cargo through hostile domains. GFP 0 s strength lies in its small size, formidable stability, and relative ease of use. It requires only oxygen and an energy source to do its work, which can be supplied at low cost and high precision, respectively. With such a low threshold for use, GFP is often the first line of inquiry into an unknown space. Here is provided a brief compendium of GFP 0 s contributions to biotechnology. They are linked by a need for a level of information that was previously inaccessible, both spatially and temporally. Protein fusions, transcriptional reporters, whole-organism visualization, and various other screening applications are reviewed with respect to biotechnological applications. Germane molecular improvements to GFP are also discussed.
Journal of Biotechnology, 2005
This study has investigated the expression of green fluorescent protein (GFP) variants in the cytosol and the endoplasmic reticulum (ER) of HeLa cells and evaluated the effects of the different cellular environments on the fluorescence properties of these GFP variants. Several GFP variants have been constructed by adding different N-or C-terminal signal sequences. These proteins were expressed and folded in distinct cellular compartments in HeLa cells. The localization of these GFP variants targeted to the endoplasmic recticulum was confirmed by the co-localization of DsRed2-ER as assessed by confocal microscopy. The addition of signal peptides targeting GFP variants to the ER or cytosol did not appear to alter the optical spectra of these GFP variants. However, the fluorescence intensity of these GFP variants in the ER was significantly less than that in the cytosol. Thus, the results clearly suggest that the cellular environment affects the formation and/or maturation of green fluorescence protein in vivo. These findings will be helpful in the future development and application of GFP technology aimed at investigating cellular functions performed in the ER and the cytosol.
From Jellyfish to the Nobel Prize: The Discovery and Uses of Green Fluorescent Protein
Green Fluorescent Protein (GFP) was first isolated from light-producing jellyfish in the 1960s. Since the first reported expression of GFP into living organisms in 1994, it has become one of the most studied proteins in biochemistry and molecular biology. The ability of fluorescent proteins to fluoresce after expression in living cells has afforded researchers a tool to track the location and trafficking of proteins in cells and intact organisms. In 2008, the Nobel Prize in Chemistry was awarded to Shimomura, Chalfie and Tsien for their contribution to the discovery and development of GFP. This review traces the key events in that discovery process and highlights some significant applications of fluorescent proteins reported worldwide, including examples from Malaysia. Innovative applications of fluorescent proteins may help to answer many current biological questions and accelerate the development of tools to prevent or treat disease.
Green fluorescent protein was initially isolated from the jellyfish Aequorea victoria. It has a unique β-barrel structure and it generates an internal fluorophore formed by an autocatalytic process. Since the decoding of the gene, GFP with its different spectral variants, became one of the most studied and used proteins in molecular biology, cell biology and medicine, as a marker of gene expression and for protein targeting in living tissues.
Long-Term, Stable Expression of Green Fluorescent Protein in Mammalian Cells
Biochemical and Biophysical Research Communications, 1997
temperature (up to 65ЊC), pH11, 1% SDS (sodium dode-Despite the proven utility of green fluorescent procyl sulphate), 6 M guanidinium chloride and remains tein (GFP) as a reporter molecule for transient gene resistant to most proteases for hours. Photobleaching expression, the adequacy of this marker for models of wild-type GFP is reportedly about half that of fluorequiring durable, high-level gene expression has rescein, and the protein's quantum yield is about 0.8. not been fully tested. To address this issue, we per-The utility of this protein in experimental biology is formed the transfection of Chinese Hamster Ovary being defined in a variety of cells. For example, GFP (CHO) cells with plasmid DNA encoding both GFP was used to track the cellular movements in the slime and neomycin phosphotransferase (neo) cassettes. mold Dictyostelium discoideum in real time (3). GFP The expression of GFP was measured after the cells fusion proteins like GFP-Lac I have been used to moniwere cultured in the presence or absence of G418tor chromosomal segregation in live bacteria (4). GFP mediated selective pressure. After removal of G418 has also been used to assess gene transfer techniques from the growth medium, the percentage of pooled (5, 6). While the merits of this marker are extensive, G418 resistant transfectants which co-expressed the some limitations have also been reported (7). GFP transgene increased or remained unchanged. Numerous publications (8-10) have proven the use-Flow cytometric and visual isolation of GFP-expressing cells was possible without continued selection in fulness of GFP as reporter molecule in the setting of G418. One cloned cell line, C463, maintained high-transient gene expression. However, it remains unclear level green fluorescence for 18 weeks in G418 and whether cell lines are able to maintain high-level GFP an additional 12 weeks in nonselective medium. Our expression over many passages in the absence of selecdata suggest expression of GFP does not confer a tive growth conditions. Here we demonstrate visual growth disadvantage in mammalian cells. ᭧ 1997 and flow cytometric isolation of mammalian cells which Academic Press maintain high-level GFP expression for months during and after removal of G418. MATERIALS AND METHODS Green fluorescent protein (GFP) is a convenient reporter molecule to monitor gene and protein expression Cell lines and transfections. Chinese Hamster Ovary (CHO) cells in a broad spectrum of model organisms (1). GFP is were grown in HAMS cell media supplemented with 10% fetal bovine able to produce green fluorescence when exited with a serum (FBS; Biofluids; Rockville, MD) and gentamicin 25mg/ml (Life blue light. No additional substrates are required to de-Technologies; Gaithersburg, MD). Cells were plated at 50% confluence in 100mm dishes. Calcium phosphate:DNA transfections were tect GFP and it can be monitored in live cells (e.g. performed according to the manufacturer's protocol (Promega; Madiprotein localization). Most GFPs currently in use are son, WI). 15mg of purified DNA (Qiagen; Chatsworth, CA) was used derived from the Pacific Northwest jellyfish, Aequorea for each transfection, and G418 (Life Technologies; Gaithersburg, victoria (2). Aequorea GFP is relatively small polypep-MD) was added to the cell media 48 hours post transfection at a final tide consisting of 238 amino acid residues. The purified, concentration of 0.7 mg/ml. properly folded GFP exhibits remarkable fluorescent Constructions of expression vectors. PCR amplification and TA stability toward different denaturants such as high cloning (Invitrogen, San Diego, CA) were used to replace the CD4 domain of pJM48 (11) with the commercially available EGFP gene (Clontech; Palo Alto, CA) and a stop codon. The resulting vector, pLCB38, encoded both EGFP and neomycin phosphotransferase 1 Address correspondence to J. L. Miller, Laboratory of Chemical Biology, Building 10, Room 9N308, National Institutes of Health, genes between adeno-associated virus inverted terminal repeats (AAV ITRs). For comparison, a plasmid without AAV ITR regions,
EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion
Proceedings of The National Academy of Sciences, 2004
A gene encoding a fluorescent protein from the stony coral Lobophyllia hemprichii has been cloned in Escherichia coli and characterized by biochemical and biophysical methods. The protein, which we named EosFP, emits strong green fluorescence (516 nm) that changes to red (581 nm) upon near-UV irradiation at Ϸ390 nm because of a photo-induced modification involving a break in the peptide backbone next to the chromophore. Single-molecule fluorescence spectroscopy shows that the wild type of EosFP is tetrameric, with strong Fö rster resonance coupling among the individual fluorophores. We succeeded in breaking up the tetramer into AB and AC subunit dimers by introducing the single point mutations V123T and T158H, respectively, and the combination of both mutations yielded functional monomers. Fusion constructs with a variety of proteins were prepared and expressed in human cells, showing that normal biological functions were retained. The possibility to locally change the emission wavelength by focused UV light makes EosFP a superb marker for experiments aimed at tracking the movements of biomolecules within the living cell.