Sunday, 1 April 2012

Green Fluorescent Protein


Green Fluorescent Protein





In 1960, Osamu Shimomura first began studying the bioluminescence of the jellyfish, Aequorea Victoria. (Zimmer 2011) By 1962 Shimomura and Frank Johnson had first isolated a calcium-dependent bioluminescent protein from the jellyfish and named it aequorin. This protein had blue-  emitting bioluminescent properties. Whilst isolating this protein Shimomura and Johnson also observed a second protein which lacked the blue-emitting bioluminescent properties of aequorin, however was able to produce green fluorescence when illuminated with blue ultraviolet-light. (Piston 2004) This was named green fluorescent protein (GFP). In 1992 the GFP gene was first cloned, with the importance of this development not realised until the mid 1990s when the GFP's properties were first used to track gene expression in bacteria and nematodes. (Piston 2004) Only within recent years has the discovery of this tracking ability been fully appreciated, with Osamu Shimomura, Martin Chalfie and Roger Tsien being award the 2008 Nobel Prize in chemistry for their discovery and development of the green fluorescent protein. (Zimmer 2011) 

The principal chromophor, the part of an organic molecule which provides it with colour, of the GFP is derived from a triplet of adjacent amino acids: serine, tyrosine and glycine residues at positions 65-67 on the sequence (Ser65, Tyr66 and Gly67 respectively). The reason for this chromophor's fluorescence is the location of this peptide triplet within the centre of a stable barrel structure, consisting of 11 beta-sheets folded into a tube. A reaction occurs between Ser67 and Gly67 resulting in the formation of an imidazolin-5-one heterocyclic nitrogen ring system. Further oxidation results in conjugation of the imidazolin ring with Tyr66, allowing the absorption of blue ultraviolet-light. Upon absorption of the blue ultraviolet-light, the chromophor gives off a green light. (Piston 2004) 

The fluorescent properties of the GFP have the potential to be hugely useful in modern science. Due to recent advances in modern technology the GFP can be easily isolated and, through transfection, be inserted into mammalian cells. This allows scientists to transfect cancer cells or donated organ cells and track their movements within mammals through the application of blue ultraviolet-light.  The process of transfection and isolation of the GFP is as follows. Mutated bacterial cells, called competent cells, which are susceptible to plasmid replication and have been chemically permeabilized to allow the transfer of DNA across the membrane and cell wall through transformation. After transformation the bacteria are matured to exponential phase, where they most rapidly reproduce, thus also replicating the DNA. The bacterial membrane is then ruptured through lysis. Lysis is simply the process of disintegrating or puncturing a cellular membrane. (Piston 2004)  The alkaline detergent solution used to rupture the membrane also contains enzymes which degrade any contaminating RNA. The lysate is then filtered and placed on an ion exchange column. All unwanted RNA, DNA and proteins are then cleared from the column. The remaining plasmid DNA is eluted using a high salt buffer. Isopropanol precipitation then concentrates the eluted plasmid DNA which is collected through centrifugation, washed and redissolved in buffer. (Piston 2004)  It is at this stage that the DNA may be used for transinfection.  

GFP's ability to be transferred into mammalian cells whilst retaining its fluorescent properties has recently been simplified through transfection. The applications of this advance in genetic modification are near limitless, but currently the goals include tracking of cancerous cells within humans and the tracking of cells transferred between humans through organ transplants. The green fluorescence is almost wholly due to a chromophor consisting of serine, tyrosine and glyscine.











References



ñ  Campbell, RE 2008, Fluorescent proteins, viewed 18/03/12 <http://www.scholarpedia.org/article/Fluorescent_proteins>

ñ  Chalfie, M, 1995, 'Green Fluorescent Protein', Photochemistry and Photobiology, Vol. 62, No. 4, pp. 651-656.

ñ  Hara, M,  et al 2002, 'Transgenic mice with green fluorescent protein-labeled pancreatic beta-cells', AJP – Endocrinology and Metabolism, pp. E177-E183.

ñ  Piston, DQ, et al 2004, Introduction to Fluorescent Proteins, viewed 18/03/12 <http://www.microscopyu.com/articles/livecellimaging/fpintro.html>

ñ  Zimmer, M 2011, Green Fluorescent Protein, viewed 18/03/12 <http://gfp.conncoll.edu/GFP-1.htm>

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