Editing Green fluorescent protein (section)

=== Wild-type GFP (wtGFP) ===

In the 1960s and 1970s, GFP, along with the separate luminescent protein [[aequorin]] (an [[enzyme]] that catalyzes the breakdown of [[luciferin]], releasing light), was first purified from the jellyfish ''Aequorea victoria'' and its properties studied by [[Osamu Shimomura]].<ref name=Shimomura_1962>{{cite journal | vauthors = Shimomura O, Johnson FH, Saiga Y | title = Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea | journal = [[Journal of Cellular and Comparative Physiology]] | volume = 59 | issue = 3 | pages = 223–39 | date = Jun 1962 | pmid = 13911999 | doi = 10.1002/jcp.1030590302 }}</ref>  In ''A. victoria'', GFP fluorescence occurs when [[aequorin]] interacts with [[calcium|Ca<sup>2+</sup>]] ions, inducing a blue glow. Some of this luminescent energy is transferred to the GFP, shifting the overall color towards green.<ref name=Morise_1974>{{cite journal | vauthors = Morise H, Shimomura O, Johnson FH, Winant J | title = Intermolecular energy transfer in the bioluminescent system of Aequorea | journal = [[Biochemistry (journal)|Biochemistry]] | volume = 13 | issue = 12 | pages = 2656–62 | date = Jun 1974 | pmid = 4151620 | doi = 10.1021/bi00709a028 }}</ref> However, its utility as a tool for molecular biologists did not begin to be realized until 1992 when [[Douglas Prasher]] reported the cloning and nucleotide sequence of wtGFP in ''Gene''.<ref name=Prasher_1992>{{cite journal | vauthors = Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ | title = Primary structure of the Aequorea victoria green-fluorescent protein | journal = [[Gene (journal)|Gene]] | volume = 111 | issue = 2 | pages = 229–33 | date = Feb 1992 | pmid = 1347277 | doi = 10.1016/0378-1119(92)90691-H }}</ref>  The funding for this project had run out, so Prasher sent [[cDNA]] samples to several labs. The lab of [[Martin Chalfie]] expressed the coding sequence of wtGFP, with the first few amino acids deleted, in heterologous cells of ''[[Escherichia coli|E. coli]]'' and ''[[Caenorhabditis elegans|C. elegans]]'', publishing the results in ''[[Science (journal)|Science]]'' in 1994.<ref name=Chalfie_1994>{{cite journal | vauthors = Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC | s2cid = 9043327 | title = Green fluorescent protein as a marker for gene expression | journal = [[Science (journal)|Science]] | volume = 263 | issue = 5148 | pages = 802–5 | date = Feb 1994 | pmid = 8303295 | doi = 10.1126/science.8303295 | bibcode = 1994Sci...263..802C }}</ref> Frederick Tsuji's lab independently reported the expression of the recombinant protein one month later.<ref name=Inouye_1994>{{cite journal | vauthors = Inouye S, Tsuji FI | title = Aequorea green fluorescent protein. Expression of the gene and fluorescence characteristics of the recombinant protein | journal = [[FEBS Letters]] | volume = 341 | issue = 2–3 | pages = 277–80 | date = Mar 1994 | pmid = 8137953 | doi = 10.1016/0014-5793(94)80472-9 | doi-access = free | bibcode = 1994FEBSL.341..277I }}</ref>  Remarkably, the GFP molecule folded and was fluorescent at room temperature, without the need for exogenous cofactors specific to the jellyfish. Although this near-wtGFP was fluorescent, it had several drawbacks, including dual peaked excitation spectra, pH sensitivity, chloride sensitivity, poor fluorescence quantum yield, poor photostability and poor folding at {{convert|37|C}}.

The first reported crystal structure of a GFP was that of the S65T mutant by the Remington group in ''[[Science (journal)|Science]]'' in 1996.<ref name="Ormo_1996"/>  One month later, the Phillips group independently reported the wild-type GFP structure in ''Nature Biotechnology''.<ref name="Yang_1996"/> These crystal structures provided vital background on [[chromophore]] formation and neighboring residue interactions. Researchers have modified these residues by directed and random mutagenesis to produce the wide variety of GFP derivatives in use today. Further research into GFP has shown that it is resistant to detergents, proteases, [[guanidinium chloride]] (GdmCl) treatments, and drastic temperature changes.<ref name="Brejc_1997">{{cite journal | vauthors = Brejc K, Sixma TK, Kitts PA, Kain SR, Tsien RY, Ormö M, Remington SJ | title = Structural basis for dual excitation and photoisomerization of the ''Aequorea victoria'' green fluorescent protein | journal = [[Proceedings of the National Academy of Sciences of the United States of America]] | volume = 94 | issue = 6 | pages = 2306–2311 | date = March 1997 | pmid = 9122190 | pmc = 20083 | doi = 10.1073/pnas.94.6.2306 | bibcode = 1997PNAS...94.2306B | doi-access = free }}</ref>