Green Fluorescent Protein
(GFP) is a ~27 kDa in size and was isolated originally from the jellyfish Aequoria victoria
. It has an endogenous fluorochrome activity with excitation maximum at 395 nm and emission maximum at 509 nm, which is similar to fluorescein (1). As a result GFP can readily be visualized using a regular fluorescence microscope. GFP fluorescence develops as soon as the protein is expressed and requires only molecular oxygen and no other cofactors. As a result GFP can be expressed in fluorescent form in essentially any prokaryotic or eukaryotic cell, so long as oxygen is present. GFP has been engineered to produce a vast number of variously colored mutants including blue, cyan and yellow protein derivatives, (BFP, CFP and YFP), which may include a variety of other properties (see 2-4). Homologues of GFP are found in other Cnidaria, and some of these produce red fluorescent proteins. GFP and its relatives are widely used as fluorescent tracers in transfection and transgenic experiments, to monitor gene expression and protein localization in vivo
. The discovery of GFP, the first demonstrations of the of the utility of this protein and the development of a variety of modified forms of GFP were honored by the 2008 Nobel Prize in Chemistry
, awarded to Osamu Shimomura, Martin Chalfie and Roger Tsien..
His-tagged recombinant aceGFP was run out on an SDS-PAGE gel at 10, 5, 2.5 µg in the right most three lanes. BSA was also run at 10 µg, 7.5 µg, 5 µg and 2.5 µg as indicated. The vector adds a C-terminal His-tag which was used to purify the protein and this, along with some other vector derived sequence adds about 5 kDa to the molecule, which therefore runs at about 30 kDa. The lane on the left contains Biorad SDS-PAGE molecular weight standards of the indicated size in kiloDaltons.
The original A. victoria GFP was engineered extensively in the Tsien lab to prevent it from forming tetramers and dimers and to modify and improve the spectral properties (1-3). The sequence of our GFP is based on a very similar GFP originally cloned out of Aequoria coerulescens (5), a jellyfish closely related to if not identical to A. victoria. The A. coerulescens protein is called acGFP and is 92% identical to the original A. victoria GFP, but perhaps surprisingly, due to a single amino acid substitution, it is not fluorescent (5). The acGFP protein was engineered to produce a strongly fluorescent form which also behaves as a monomer in solution and which is referred to as aceGFP, the exact sequence being deposited here. The aceGFP, is expressed in several widely used commercial expression vectors such as pTRE-G1, pAcGFP1-C, pIRES2-AcGFP1, pT7XbG2-AcGFP1 and pcDNA6.2/C-EmGFP-DEST, some of which are currently marketed by Invitrogen and other companies. We generated a cDNA encoding aceGFP and expressed it in and purified it from E. coli as shown in the figure. The expression vector pET29a adds an C-terminal His-tag which was use to purify the protein and this, along with some other vector derived sequence, adds about 5 kDa to the molecule. The construct therefore has a total size of about 30 kDa as shown.
1. Baird GS, Zacharias DA, Tsien RY. Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci U S A. 97:11984-9 (2000).
2. Gross LA, Baird GS, Hoffman RC, Baldridge KK, Tsien RY. The structure of the chromophore within DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci U S A. 97:11990-5 (2000).
3. Heikal AA, Hess ST, Baird GS, Tsien RY, Webb WW. Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: coral red (dsRed) and yellow (Citrine). Proc Natl Acad Sci U S A. 97:11996-2001 (2000).
4. Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature Biotechnology 22:1567-1572 (2004).
5. Gurskaya, NG, Fradkov AF, Pounkova, NI, Staroverov DB, Bulina, ME, Yanushevich YG, Labas YA, Lukyanov S and Lukyanov, KA. A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants. Biochem. J. 373, 403–408 (2003)
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