GREEN FLUORESCENT PROTEIN TAGGING

GREEN FLUORESCENT PROTEIN

INTRODUCTION Green fluorescent protein - GFP is a naturally fluorescent protein that has been present in a species of deep sea jelly fish -Aequorea Victoria for nearly two hundred million years and its responsible for the bioluminescence of this jelly fish. Osamu Shimomura isolated GFP in 1962 and identified the component of GFP responsible for its fluorescence. His work laid the foundation on which the GFP revolution was built (Shimomura et al., 1962). GFP is a 238 aminoacid protein with a molecular weight of 27 kDa. It has a unique canlike shape of 11 strand -barrel with a single alpha helical strand containing the chromophore running through the center. This structure allows chromophore formation and protects it from the surrounding microenvironment (Yang et al., 1996). PROCESS OF FLUORESCENCE When exposed to blue light GFP fluoresces green (Tsien, 1998). In A. victoria, GFP fluorescence occurs when the luminescent protein aequorin interacts with Ca2+ ions. This interaction induces a blue glow and some of this energy is transferred to the protein thereby shifting the overall color towards green [figure 1] (Morise, 1974).

FIGURE 1: PROCESS OF FLUORESCENCE OF GFP IN JELLY FISH

(Zimmer M., Glowing Genes) However, its utility as a tool for molecular biologists was not realized until Douglas Prasher in 1992 (Woodshole Oceanographic Institution) cloned and reported the nucleotide sequence of GFP in Gene (Prasher et al., 1992). This paved the way for initial experiments in protein tagging by Tulle Hazelrigg of Columbia University and its expression in the nematode worm - Caenorhabditis elegans by Martin Chalfie, also at Columbia. Martin Chalfie was the first to use GFP for labeling in 1994 (Chalfie et al., 1994) after which, Roger Y. Tsien and others have lead the way in vivo labeling of proteins for monitoring the expression, localization, and transportation of tagged proteins in living cells. The success of this method is remarkable despite the fact that GFP adds an additional 238 residues to the N or C terminus of the fusion protein (Zimmer, 2005).

FIGURE 2: INDRODUCTION OF GFP SEQUENCE INTO THE GENE OF THE PROTEIN OF

INTEREST AND ITS EXPRESSION. (Zimmer M., Glowing Genes)

STRUCTURE RESPONSIBLE FOR FLUORESCENCE The chromophore (Figure 3) of GFP is responsible for its fluorescence. The chromophore is located in the middle of the beta-barrel; it is occasionally referred to as the light in the can (Figure 4). GFP catalyzes the formation of its own chromophore. It is proposed that Arg96 plays a crucial role in this catalysis process.

FIGURE 3: STRUCTURE OF THE CHROMOPHORE OF GFP (where R groups are the first 64 and last 170 residues of GFP) (Zimmer M., Glowing Genes)

Now GFP is found in labs all over the world where it is used in many plant and animal species. The green fluorescence protein gene is frequently used as a reporter of expression (Phillips, 2001) and in modified forms to make biosensors. Many animals have been created that express GFP. Researchers have modified the structure of GFP protein by directed and random mutagenesis of its residues to produce the wide variety of GFP derivatives which are in use today.

PROCESS OF TAGGING: Tagging proteins with GFP is very simple where the gene for GFP is attached to one end of the gene that encodes the protein of interest (Figure 4). Similar to other tagging methods this gene can also be fused with the gene of interest. The recombinant DNA encodes a chimeric protein that contains the property of both proteins (it performs the function of the gene of interest and at the same time can exhibit fluorescence). In most cases, the GFP fusion protein behaves just like the original protein and the movement of the proteins can then be monitored by following its fluorescence inside the cell using fluorescence microscopy.

FIGURE 3: FLOWCHART OF THE PROCESS OF GFP TAGGING (Flow chart from www.majorgrooves.co.uk/gfp.html)

It takes an hour or so for the translated protein to exhibit fluorescence after the addition of GFP by undergoing self-catalyzed post translational modification to create an efficient and bright fluorescent center which is shielded within the interior of a barrel shaped protein. ADVANTAGES OF USING GFP AS A TAG PROTEIN: GFP is a well characterized protein. Its sidechain contains the residues serine, threonine and glycine which spontaneously react with one another to form a fluorescent chromophore. This is distinct from other bioluminescent proteins used as tags as it does not require cofactors, substrates or any other additional proteins and is extremely stable. The GFP gene can be introduced into organisms with local injection and maintained in their genome through breeding.

Most small fluorescent molecules such as fluorescein isothiocyanate (FTC) are strongly phototoxic when used in live cells, fluorescent proteins such as GFP are usually much less harmful when illuminated in living cells. This has triggered the development of highly automated live cell fluorescence microscopy systems which can be used to observe cells express fluorescent proteins over-time. These studies with time lapse have redefined the understanding of many biological processes. GFP and its derivatives have redefined fluorescence microscopy and its uses in biology (Yuste, 2005). The fact that GFP requires no additional substrates and be monitored non-invasively in living cells has been a revolutionary advance. It can also be used as a quantitative marker in living cells due to the many mutations in wild type GFP. It can also be used as a tag for studying localisation and movement of protein-GPF chimeras within cells in real time (Chudakov, 2005).