Fluorescent Proteins:
Bioluminescence is used as a defense, offense and communication mechanism by various organisms in nature including insects, fish, squid, sea cacti, sea pansies, clam, shrimps and jellyfish(1). After the discovery of the Green Fluorescent Protein (GFP) by Shimomura et al. from Aequorea jellyfish(2), an unprecedented tool for visualizing living organisms was also being introduced to molecular biology, leading to a Nobel Prize in 2008(3). The Aequorea GFP is a 238 aa, 30 kDa monomer that emits green fluorescence maximum at 509 nm wavelength(4). It has a unique structure with an 11 stranded β - barrel like shape involving α- helixes running through the center of the can; to which the chromophore is attached(5). Autocatalytic formation of the chromophore of the GFP without the neccessity of any substrates or cofactors is a rather useful property of the GFPs allowing them to express in fusion with different proteins. Remarkably, the fusion of GFP to a protein does not have any vital effect on the activity or mobility of the protein in addition to its nontoxic nature(1).
Figure 1: Structure of the GFP taken from the PDB, code 1EMA
References:
- (1) Zimmer, M. (2002) “Green Fluorescent Protein (GFP): Applications, Structure and Related Photophysical Behavior.” Chemical Reviews. v. 102 (3) pp. 759-781.
- (2) Shimomura, O., Johnson, F.H. & Saiga, Y. (1962) “Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan.” Aequorea. J. Cell. Comp. Physiol. v. 59, pp. 223–239.
- (3) Shimomura, O., Chalfie, M. & Tsien, R. Y. (2008) “The discovery and development of the green fluorescent protein, GFP.” The Nobel Prize in Chemistry. Retrieved from the website http://nobelprize.org/nobel_prizes/chemistry/laureates/2008/
- (4) Prasher, D., C., Eckenrode, V., K., Ward,W.,W., Prendergast, F., G. & Cormier, M., J. (1992) “Primary structure of the Aequorea victoria green-fluorescent protein” Gene., v. 111, pp. 229-233.
- (5) Tsien, R., Y. (1998) “The Green Fluorescent Protein” Annual Review of Biochemistry. v. 67, pp. 509–544
Plasmid backbone:
pSP-GM1
SNF1 (modified subunits):
Snf1 (alfa), Snf4 (gamma)
FP positions:
N-terminal alfa with N-terminal gamma, N-terminal gamma with C-terminal gamma
Primer design:
Fusion primers with restiriction enzyme sites included at very ends, each annealing part was desinged with an melting temperature of around 60 C
|
"
