GFP
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| - | *[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=1169893 wtGFP on NCBI | + | ==General Info== |
| - | + | [[Image:GFP structure.png|thumb|200px|GFP ribbon diagram. From [http://www.rcsb.org/pdb/explore/explore.do?structureId=1EMA PDB 1EMA].]] | |
| - | The | + | * Originally discovered and characterized in the jellyfish ''Aeqorea victoria'' |
| + | *[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=1169893 wtGFP sequence] on NCBI | ||
| + | * 238 amino acids | ||
| + | * The tertiary structure is a fluorophore [Ser(65)-Tyr(66)-Gly(67)] nestled inside a protective beta barrel. | ||
| + | * Oxygen is required for maturation of the fluorophore | ||
| + | * Natural state is a dimer with a Kd of ~ 100 µM. | ||
==GFP Variants== | ==GFP Variants== | ||
| - | *GFPmut3b - S65G, S72A | + | * '''GFPmut2''' - S65A, V68L, S72A <cite>Cormack</cite> |
| - | *[http:// | + | * '''GFPmut3b''' - S65G, S72A <cite>Cormack</cite> |
| - | **This is GFPmut3b | + | *[http://partsregistry.org/wiki/index.php/Part:BBa_E0040 '''''GFP''mut3*'''] - mutations from wt: S2R,S65G,S72A |
| - | *[[Emerald]] | + | **This is GFPmut3b with an accidental mutation at position 2 that doesn't affect function according to the authors.<cite>Andersen</cite> |
| - | * EGFP F64L, S65T + optimized for human codons <cite>li97</cite> | + | *'''[[Emerald]]''' - F64L, S65T, S72A, N149K, M153T, I167T |
| + | * '''EGFP''' - inserted GTG as second codon, F64L, S65T + optimized for human codons with 35-fold increase in fluorescence over GFP <cite>li97</cite> | ||
| + | ** Additionally, "mutation of His231 to Leu, which was probably inadvertent and neutral". <cite>li97</cite> | ||
| + | * '''yemGFP''' - F64L, S65T, A206K <cite>Stricker08</cite> | ||
| + | * [http://partsregistry.org/Part:BBa_I746916 '''superfolder GFP'''] - S30R, Y39N, F64L, (S65T/G65T), F99S, N105T, Y145F, M153T, V163A, I171V, A206V <cite>Pedelacq06</cite> | ||
| + | ** Inherited parent variant mutations (GFPmut3*): S2R, <s>S65G</s>, S72A (S65G mutation is superseded by S65T mutation, hence the alternative mutation designation of G65T) | ||
| + | * Other variants and their mutations can be found in Shaner supplementary table 2 <cite>shaner05</cite> | ||
| + | |||
| + | ==Mutations== | ||
| + | * '''F64L''': improve folding @37C | ||
| + | * '''S65T''': 5-6x increase in amplitude and red shift | ||
| + | * '''S65(G|T) and T203(Y|F|W|H)''': GFP -> YFP | ||
| + | * '''Y66W''': GFP -> CFP | ||
| + | * '''Y66H''': GFP -> BFP (more blue than CFP), dim, easily photobleached | ||
| + | * '''Y66F''': excitation 360nm, emission 442nm | ||
| + | * '''R96A''': ''slows'' cyclization reaction from minutes to months | ||
| + | * '''F99S, M153T, V163A''': "cycle 3" mutations (originally reported as F100S, M154T, V164A) <cite>Crameri96</cite> | ||
| + | * '''Y203I''': eliminate excitation peak at 475nm, leaving lower peak of 399nm. emission remains at 511nm, producing large Stokes shift | ||
| + | * '''A206K''': make monomeric | ||
| + | * '''S30R, Y39N, N105T, Y145F, I171V, A206V''': additional superfolder mutations <cite>Pedelacq06</cite> | ||
==References== | ==References== | ||
| + | |||
| + | ===General=== | ||
<biblio> | <biblio> | ||
| + | #prasher pmid=1347277 | ||
| + | //original cloning of GFP | ||
| + | #chalfie pmid=8303295 | ||
| + | //original use of GFP as a reporter | ||
| + | #tsien98 pmid=9759496 | ||
| + | //good review of GFP | ||
#li97 pmid=9353317 | #li97 pmid=9353317 | ||
#Cormack pmid=8707053 | #Cormack pmid=8707053 | ||
#Andersen pmid=9603842 | #Andersen pmid=9603842 | ||
| + | #zacharias [http://www.tsienlab.ucsd.edu/Publications/Zacharias%202006%20-%20Molecular%20Biology%20and%20Mutation%20of%20GFP.pdf Molecular Biology and Muation of Green Fluorescent Protein (Book Chapter, Zacharias & Tsien)] | ||
| + | #shaner05 pmid=16299475 | ||
| + | #Stricker08 pmid=18971928 | ||
| + | #Pedelacq06 pmid=16369541 | ||
| + | #Crameri96 pmid=9630892 | ||
| + | </biblio> | ||
| + | |||
| + | ===GFP as a measure of gene expression=== | ||
| + | <biblio> | ||
| + | #Lindow pmid=11698362 | ||
| + | #Iafolla pmid=18350571 | ||
| + | #Bagh pmid=18352063 | ||
| + | #Dong pmid=18352052 | ||
</biblio> | </biblio> | ||
| - | [ | + | [[Category:Material]] [[Category:Protein]] [[Category:Reporter]] |
Current revision
Contents |
General Info
- Originally discovered and characterized in the jellyfish Aeqorea victoria
- wtGFP sequence on NCBI
- 238 amino acids
- The tertiary structure is a fluorophore [Ser(65)-Tyr(66)-Gly(67)] nestled inside a protective beta barrel.
- Oxygen is required for maturation of the fluorophore
- Natural state is a dimer with a Kd of ~ 100 µM.
GFP Variants
- GFPmut2 - S65A, V68L, S72A [1]
- GFPmut3b - S65G, S72A [1]
- GFPmut3* - mutations from wt: S2R,S65G,S72A
- This is GFPmut3b with an accidental mutation at position 2 that doesn't affect function according to the authors.[2]
- Emerald - F64L, S65T, S72A, N149K, M153T, I167T
- EGFP - inserted GTG as second codon, F64L, S65T + optimized for human codons with 35-fold increase in fluorescence over GFP [3]
- Additionally, "mutation of His231 to Leu, which was probably inadvertent and neutral". [3]
- yemGFP - F64L, S65T, A206K [4]
- superfolder GFP - S30R, Y39N, F64L, (S65T/G65T), F99S, N105T, Y145F, M153T, V163A, I171V, A206V [5]
- Inherited parent variant mutations (GFPmut3*): S2R,
S65G, S72A (S65G mutation is superseded by S65T mutation, hence the alternative mutation designation of G65T)
- Inherited parent variant mutations (GFPmut3*): S2R,
- Other variants and their mutations can be found in Shaner supplementary table 2 [6]
Mutations
- F64L: improve folding @37C
- S65T: 5-6x increase in amplitude and red shift
- S65(G|T) and T203(Y|F|W|H): GFP -> YFP
- Y66W: GFP -> CFP
- Y66H: GFP -> BFP (more blue than CFP), dim, easily photobleached
- Y66F: excitation 360nm, emission 442nm
- R96A: slows cyclization reaction from minutes to months
- F99S, M153T, V163A: "cycle 3" mutations (originally reported as F100S, M154T, V164A) [7]
- Y203I: eliminate excitation peak at 475nm, leaving lower peak of 399nm. emission remains at 511nm, producing large Stokes shift
- A206K: make monomeric
- S30R, Y39N, N105T, Y145F, I171V, A206V: additional superfolder mutations [5]
References
General
- Cormack BP, Valdivia RH, and Falkow S. . pmid:8707053.
- Andersen JB, Sternberg C, Poulsen LK, Bjorn SP, Givskov M, and Molin S. . pmid:9603842.
- Li X, Zhang G, Ngo N, Zhao X, Kain SR, and Huang CC. . pmid:9353317.
- Stricker J, Cookson S, Bennett MR, Mather WH, Tsimring LS, and Hasty J. . pmid:18971928.
- Pédelacq JD, Cabantous S, Tran T, Terwilliger TC, and Waldo GS. . pmid:16369541.
- Shaner NC, Steinbach PA, and Tsien RY. . pmid:16299475.
- Crameri A, Whitehorn EA, Tate E, and Stemmer WP. . pmid:9630892.
- Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, and Cormier MJ. . pmid:1347277.
original cloning of GFP - Chalfie M, Tu Y, Euskirchen G, Ward WW, and Prasher DC. . pmid:8303295.
original use of GFP as a reporter - Tsien RY. . pmid:9759496.
good review of GFP - Molecular Biology and Muation of Green Fluorescent Protein (Book Chapter, Zacharias & Tsien)
GFP as a measure of gene expression
- Leveau JH and Lindow SE. . pmid:11698362.
- Iafolla MA, Mazumder M, Sardana V, Velauthapillai T, Pannu K, and McMillen DR. . pmid:18350571.
- Bagh S, Mazumder M, Velauthapillai T, Sardana V, Dong GQ, Movva AB, Lim LH, and McMillen DR. . pmid:18352063.
- Dong GQ and McMillen DR. . pmid:18352052.
