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us your comments or suggestions. Updated August 12, 1998
Green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a revolutionary reporter molecule for monitoring gene expression and protein localization in vivo, in situ, and in real time (1-4). GFP fluoresces bright green upon mere exposure to UV or blue lightunlike other bioluminescent reporters which require additional proteins, substrates, or cofactors to emit light. While intact GFP is required for fluorescence, the active chromophore in GFP is a cyclic tripeptide (5) encoded in the primary amino acid sequence. Chromophore formation is oxygen dependent, occurs gradually after translation (6), and does not appear to be enzymatic (7).
GFP fluorescence is stable, species independent, and can be monitored noninvasively in living cells. GFP fluorescence persists in formaldehyde-fixed cells and is well suited for double-labeling experiments with other fluorescent markers (including the GFP variants described below; 8). GFP has been expressed and shown to fluoresce in a variety of species (see the GFP Bibliography for a list).
CLONTECH offers several optimized GFP variants, including EGFP (GFPmut1; 8-9), EBFP, EYFP, d2EGFP, ECFP, and GFPuv (10). EGFP encodes a protein which has a single, red-shifted excitation peak and fluoresces about 35 times more intensely than wt GFP when excited at 488 nm (11), due to an increase in its extinction coefficient (Em). To ensure maximal mammalian expression, the coding region of EGFP contains more than 190 silent base mutations which correspond to human codon-usage preferences (11). The red-shifted spectrum and increased expression of EGFP make it ideal for fluorescence microscopy and fluorescence-activated cell sorting (FACS; 8, 12).
Destabilized EGFP† (d2EGFP) contains the PEST domain from mouse ornithine decarboxylase (MODC), fused to the C-terminus of EGFP (13). This domain targets EGFP for rapid turnover, effectively reducing EGFP's half-life to two hours. The introduction of d2EGFP greatly increases the utility of GFP in studying dynamic cellular events in vivo.
Enhanced cyan fluorescent protein (ECFP) contains six mutations that result in cyan fluorescence. The coding sequence of ECFP has also been codon optimized for higher expression in mammalian cells. ECFP has fluorescence excitation and emission maxima similar to other cyan emission variants (7, 15, 17). ECFP photobleaches less rapidly than EBFP and is useful in double- and triple-labeling experiments with EGFP and/or EYFP. ECFP may also be used in FRET (fluorescent resonance energy transfer) experiments for co-expression with another fluorescent protein.
Enhanced blue fluorescent protein (EBFP) contains four amino acid substitutions that allow the protein to emit blue light. The coding sequence of EBFP has also been codon optimized for higher expression in mammalian cells. This blue variant is 23-fold brighter than other blue variants (14) and photobleaches one-half to one-third less quickly than P4-3 (15), a popular predecessor to EBFP.
Enhanced yellow fluorescent protein (EYFP) contains mutations (GFP-10C; 16) that shift the emission of the GFP chromophore from green to yellowish-green. EYFP is also human codon optimized for higher expression in mammalian systems, and fluorescence is roughly equivalent to that of EGFP.
GFPuv was developed at Maxigen by Crameri et al. (10) and is optimized for maximal fluorescence when excited by UV light (360-400 nm). GFPuv contains three amino acid substitutions which make E. coli expressing GFPuv fluoresce 18 times brighter than wt GFP. Additionally, five Arg codons from the wt GFP gene were replaced by codons preferred in E. coli, thus increasing the translational efficiency. This variant is ideal for visualizing bacterial or yeast colonies expressing GFPuv using UV light.
The different GFP variants can be used in combination for many double-labeling applications. For example, experiments can be performed by selective excitation of wt GFP and EGFP or EBFP and GFPuv, or by expressing EGFP and EBFP (or EYFP and EBFP) in the same cell. This technology has several promising applications, including microscopy of multiple cell populations in a mixed cell culture (8); monitoring gene expression from two different promoters in the same cell, tissue, or organism; monitoring the localization of two different protein fusions in the same cell, tissue, or organism; and FACS of mixed cell populations.
The Living Colors line of GFP-related products includes bacterial and mammalian expression vectors, sequencing primers, purified recombinant GFP proteins, and GFP-specific monoclonal and polyclonal antibodies. All of these products (except sequencing primers) are provided with the Living Colors User Manual (PT2040-1). This handbook contains detailed information about GFP and its variants with protocols for the expression and detection of fluorescent proteins.
For additional fluorescent protein resources, also see gfp.clontech.com and the Living Colors Vectors Comparison Table.
Please click on the links below to access the various Living Colors products.
| Living Colors Vectors (including GFP, GFPuv, EGFP, EBFP, ECFP, and EYFP) | Recombinant Fluorescent Proteins |
| Destablized EGFP Vectors | Living Colors Antibodies |
| pNeoEGFP & pHygEGFP Vectors | GFP Sequencing Primers |
References
GFP License StatementPlease send
us your comments or suggestions.
Updated August 12, 1998