Green Fluorescent Protein (GFP)

Green Fluorescent Protein (GFP) is a protein produced by a jellyfish Aequorea victoria; which produces glowing points of light around the margin of it's umbrella. The light arises from yellow tissue masses that each consist of about 6000-7000 photogenic cells. These cells generate light by a process of bioluminescence, whose components include a calcium-activated photoprotein (aequorin) that emits blue-green light and an accessory green fluorescent protein (GFP), which accepts energy from aequorin and re-emits it as green light. GFP a 238 amino acids protein which is very stable in neutral buffers up to 65oC, and displays a broad range of pH stability from 5.5 to 12. The protein is intensely fluorescent, with a quantum efficiency of approximately 80% and molar extinction coefficient of 2.2 x 104 cm-1 M-1. GFP fluoresces maximally when excited at 400nm with a lesser peak at 475nm, and fluorescence emission peaks at 509nm. The intrinsic fluorescence of the protein is due to a unique covalently attached chromophore, which is formed post-translationally within the protein upon cyclisation and oxidation of residues 65-67, Ser-Tyr-Gly. The gene for GFP has been isolated and has become a useful tool for making expressed proteins fluorescent by creating chimeric genes composed of those of GFP and its different colour variants linked to genes of proteins of interest. Making it possible to have an in vivo fluorescent protein, which may be followed in a living system.

There have been several recent developments for the use of GFP and it's colour variants. Wild type GFP has two excitation peaks, a major one at 395nm (long wave UV, causes rapid quenching of the fluorescence) and a smaller one at 475nm (blue) and an emission peak at 509nm (green). For general fluorescence microscopy purposes, investigators have been using normal FITC filter sets for viewing GFP. These are inadequate for wild type GFP both in excitation 475-495nm, and emission 520-560nm. To alleviate this problem, several modified versions of GFP were constructed which have increased fluorescence (serine to threonine substitution at position 65 increased fluorescence 5-6 times), but perhaps more important, the major excitation peak has been red-shifted to 490nm with the emission staying at 509nm. This is better for use of FITC filter sets as this modified GFP has the same excitation range as FITC. Furthermore, in confocal microscopy the main laser line used for GFP excitation is from the argon laser at 488nm, there is no good commonly used laser line near 395nm. In Arabidopsis plants and cells, poor or no fluorescence was seen when transform with gfp cDNA because the expression of GFP was curtailed by aberrant mRNA splicing. Therefore, modified forms of GFP were created to restore and improve expression of the fluorescent protein. The modified gene now contains an altered codon to remove a cryptic plant intron. Since then, other modifications have given further improvements in the brightness of the emission and different colour variants of GFP have been produced e.g. in order from shortest to longest emission spectra: blue (FP or BFP), cyan (CFP), green (GFP), yellow (YFP ) and red (RFP). This now makes it possible to make double-labelled specimens expressing two or more fluorescently labelled proteins. Added peptide sequences also allow targeting of GFP intracellular organelles like the lumen of the endoplasmic reticulum.

Useful companies for GFP research are Chroma Technology has been specializing in making single and dual label filter sets for the various forms of GFP and Clontech make the vectors for GFP and GFP colour variant for gene cloning and construction. Other useful information about GFP in plants can be found in Jim Hasselhoffs web-site at Cambridge University.