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DISCOVERY

The two best-characterized green fluorescent proteins (GFPs) are from marine invertebrates: Aequorea victoria , and a sea pansy from the Georgia coastline, Renilla reniformis [8]. Other members of this coelenterate sub-phylum Cnidaria contain fluorescent proteins which remain to be characterized. Aequorea and Renilla GFPs each transmute blue chemiluminescence from a distinct primary photoprotein into green light. In 1961 Osamu Shimomura was studying the bioluminescence of the jellyfish Aequorea at the Friday Harbor Laboratories of the University of Washington. They were trying develop a pratical method to isolate the light-emitting matter of the jellyfish, a substance later named “aequorin”. In the course of their experiments,however, they understood that the light emitted from the extract was clearly blue, contrary to their expectation of green light identical to the luminescence of live specimens. Soaking a specimen of jellyfish in a diluit potassium chloride (KCl) solution in a darkroom causes the light organs to luminescence, exhibiting a ring of bright green ring first observed, which gradually changes into blue with the progress of the cytolysis of cell. Under the ultraviolet light, a specimen of jellyfish exhibits a ring of brilliant green fluorescent, similar to the luminescence caused by KCl.

The jellyfish Aequorea

http://mekentosj.com/science/fret/images/jelly.jpg

To clarify the mechanism of energy transfert involved in the emission of green light from the jellyfish Aequorea, they isolated and purified the green fluorescent protein from the jellyfish, and then studied it's properties in detail. The purified Aequorea GFP was easily crystallized by decreasing the ionic strenght of the solvent . They investigated the energy transfer from aequorin molecule to GFP molecule during the Ca 2+ triggered luminescence reaction of aequorin, under two sets conditions: one with high concentrations of GFP (1.7-5.5 mq/ml) and the other with relatively low concentrations of GFP (0.15-1.1 mg/ml). In the presence of the high concentrations of GFP, apparently an energy transfer by the trivial (radiative) mechanism takes place, at least to some extent. The light emitted from aequorin (emission l max 460nm) is absorbed by GFP ( l max 400 and 480nm), followed by reemission of the absorbed energy from GFP as fluorescence (emission l max 509nm). In this mechanism, the extent of energy transfer and the spectral shape of emitted light are dependent on the GFP concentration. It is clear, that GFP cannot absorb all the light emitted from aequorin, because the luminescence emission of aequorin extends to about 600nm on the red side of wavelenght whereas GFP can absorb light up to only about 510nm. Therefore,a complete enerfy transfer by the trivial mechanism is clearly impossible.

http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm

When aequorin was luminesced with Ca2+ in a low ionic strenght buffer ( 10mM sodium phosphate), the emission spectrum of aequorin was little affect by GFP. Another kind of green fluorescent protein, the GFP of the sea pansy Renilla, was purified and physicochemically characterized. There are substantial differences between the bioluminescence systems of Aequorea and Renilla, thought in both system the light energy is provide by the oxidation of coelenterazine. The in vivo bioluminescence reaction of Renilla requires coelenterazine (the luciferin), Renilla luciferase, Renilla GFP, and molecular oxygen, whereas that of Aequorea requires only aequorin, Ca2+, and Aequorea GFP.

Extraction of Aequorin [9][10]

The method originally used by Shimomura was considerably modified and improved by 1970 to allow the extraction of a large number of specimens. The strips containing light organs prepared from 500 mature speciems are kepr in 800 ml of chilled seawater. The seawater is drained through a piece of Dacron gauze, and strips are added into a 2l flask containing 1l of cold 50mM EDTA,pH 6.0-6.5, saturated with ammonium sulfate, whith causes the strips to shrink. The flask is vigorously shaken for about one minute to dislodge the particles containing light organs from the strips. Then, the luminescence activity of the particles (fluorescent in green) can be also visually monitored under long-wave ultraviolet light. The mixture is squeezed through a piece of Dacron gauze. The tissue mass retained on the gauze is discared. Now, it is mixed with 80ml of analytical grade Celite powder and filtered on paper. The filter cake is then washed with EDTA. the contenents of the suction flsk are stirred occasionally for one day in a refrigerator to complete the precipitation of crude Aequorin.

As GFP has become widely used, several reports on GFP purification have emerged. The majority of these studies deal with purification of GFP alone, instead of GFP fusion proteins. The reported strategies have been tailored for particular expression systems as the background constituents largely affect the success of a scheme. To this end, most of the detailed purification studies deal with purification of recombinant GFP or its variants from Escherichia coli homogenate. Methods including hydrophobic interaction, size-exclusion, and ion-exchange chromatography, phase partitioning, organic solvent extraction, and salt and metal precipitation have all been employed, sometimes in combination, with varying degrees of success. As for GFP fusion proteins, reported purification procedures have been developed mainly based on the properties of the fusion partner (and hence difficult to extrapolate to general use with other GFP fusion proteins) or by means of an affinity tag (such as a hexa-histidine tag) . While the literature for GFP purification is abundant, no general purification protocol for GFP fusion proteins expressed in plant suspension cells has yet been reported. Plant cell culture is a potential platform for large-scale production of recombinant proteins. Purification of recombinant proteins from cultured plant cells, however, has not been widely studied. The aim of this study was to develop a general and gentle purification scheme for the recovery of GFP fusion proteins from tobacco suspension cells in high yield and purity, based mainly on the unique property of the GFP moiety. Plant cellular extracts, especially tobacco, contain phenolics that may interfere with protein recovery.

 

Purification of Aequorin [11]

The purification method of aequorin reported by Shimomura was essentially the repletion of colm chromatography on DEAE-cellulose, the only usable, efficient chromatographic adsorbent avaible at the time. Since then, various different types of chromatographic media have been developed, and the purification method has been steadily improved. The methods and techniques presently available for the purification of aequorin are summarized below. The pH is 6.5-8,and chromatography is perdormed at 0-5°C.

•  Size-exclusion chromatography (gel filtration)

•  Anion exchenge chromatography

• Hydrophobic interaction chromatography

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