Difference between revisions of "GFP"

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*[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=1169893 wtGFP sequence] on NCBI
 
*[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=1169893 wtGFP sequence] on NCBI
 
* 238 amino acids
 
* 238 amino acids
* The tertiary structure is a fluorophore [Ser(65)-Tyr(66)-Gly(67) <cite>zacharias</cite>] nestled inside a protective beta barrel.
+
* 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
 
* 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://parts.mit.edu/registry/index.php/Part:BBa_E0040 ''GFP''mut3*] - mutations from wt: S2R,S65G,S72A
+
* '''GFPmut3b''' - S65G, S72A <cite>Cormack</cite>
**This is GFPmut3b<cite>Cormack</cite> with an accidental mutation at position 2 that doesn't affect function according to the authors.<cite>Andersen</cite>
+
*[http://partsregistry.org/wiki/index.php/Part:BBa_E0040 '''''GFP''mut3*'''] - mutations from wt: S2R,S65G,S72A
*[[Emerald]] - F64L, S65T, S72A, N149K, M153T, I167T
+
**This is GFPmut3b with an accidental mutation at position 2 that doesn't affect function according to the authors.<cite>Andersen</cite>
* EGFP - added GTG as second codon, F64L, S65T + optimized for human codons with 35-fold increase in fluorescence over GFP <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, S72A (S65G mutation is superseded by S65T mutation, hence the alternative mutation designation of G65T)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>
 
* Other variants and their mutations can be found in Shaner supplementary table 2 <cite>shaner05</cite>
  
 
==Mutations==
 
==Mutations==
* F64L: improve folding @37C
+
* '''F64L''': improve folding @37C
* S65T: 5-6x increase in amplitude and red shift  
+
* '''S65T''': 5-6x increase in amplitude and red shift  
* S65(G|T) and T203(Y|F|W|H) -> YFP
+
* '''S65(G|T) and T203(Y|F|W|H)''': GFP -> YFP
* Y66W: GFP -> CFP
+
* '''Y66W''': GFP -> CFP
* Y66H: GFP -> BFP (more blue than CFP), dim, easily photobleached
+
* '''Y66H''': GFP -> BFP (more blue than CFP), dim, easily photobleached
* Y66F: excitation 360nm, emission 442nm
+
* '''Y66F''': excitation 360nm, emission 442nm
* R96A: ''slows'' cyclization reaction from minutes to months
+
* '''R96A''': ''slows'' cyclization reaction from minutes to months
* Y203I: eliminate excitation peak at 475nm, leaving lower peak of 399nm. emission remains at 511nm, producing large Stokes shift
+
* '''F99S, M153T, V163A''': "cycle 3" mutations (originally reported as F100S, M154T, V164A) <cite>Crameri96</cite>
* A206K: make monomeric
+
* '''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==
Line 41: Line 49:
 
#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)]
 
#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
 
#shaner05 pmid=16299475
 +
#Stricker08 pmid=18971928
 +
#Pedelacq06 pmid=16369541
 +
#Crameri96 pmid=9630892
 
</biblio>
 
</biblio>
  

Latest revision as of 23:15, 15 January 2013

General Info

GFP ribbon diagram. From PDB 1EMA.
  • 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)
  • 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

  1. Cormack BP, Valdivia RH, and Falkow S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene. 1996;173(1 Spec No):33-8. PubMed ID:8707053 | HubMed [Cormack]
  2. Andersen JB, Sternberg C, Poulsen LK, Bjorn SP, Givskov M, and Molin S. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl Environ Microbiol. 1998 Jun;64(6):2240-6. PubMed ID:9603842 | HubMed [Andersen]
  3. Li X, Zhang G, Ngo N, Zhao X, Kain SR, and Huang CC. Deletions of the Aequorea victoria green fluorescent protein define the minimal domain required for fluorescence. J Biol Chem. 1997 Nov 7;272(45):28545-9. PubMed ID:9353317 | HubMed [li97]
  4. Stricker J, Cookson S, Bennett MR, Mather WH, Tsimring LS, and Hasty J. A fast, robust and tunable synthetic gene oscillator. Nature. 2008 Nov 27;456(7221):516-9. DOI:10.1038/nature07389 | PubMed ID:18971928 | HubMed [Stricker08]
  5. Pédelacq JD, Cabantous S, Tran T, Terwilliger TC, and Waldo GS. Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol. 2006 Jan;24(1):79-88. DOI:10.1038/nbt1172 | PubMed ID:16369541 | HubMed [Pedelacq06]
  6. Shaner NC, Steinbach PA, and Tsien RY. A guide to choosing fluorescent proteins. Nat Methods. 2005 Dec;2(12):905-9. DOI:10.1038/nmeth819 | PubMed ID:16299475 | HubMed [shaner05]
  7. Crameri A, Whitehorn EA, Tate E, and Stemmer WP. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat Biotechnol. 1996 Mar;14(3):315-9. DOI:10.1038/nbt0396-315 | PubMed ID:9630892 | HubMed [Crameri96]
  8. Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, and Cormier MJ. Primary structure of the Aequorea victoria green-fluorescent protein. Gene. 1992 Feb 15;111(2):229-33. PubMed ID:1347277 | HubMed [prasher]
    original cloning of GFP
  9. Chalfie M, Tu Y, Euskirchen G, Ward WW, and Prasher DC. Green fluorescent protein as a marker for gene expression. Science. 1994 Feb 11;263(5148):802-5. PubMed ID:8303295 | HubMed [chalfie]
    original use of GFP as a reporter
  10. Tsien RY. The green fluorescent protein. Annu Rev Biochem. 1998;67:509-44. DOI:10.1146/annurev.biochem.67.1.509 | PubMed ID:9759496 | HubMed [tsien98]
    good review of GFP
  11. Molecular Biology and Muation of Green Fluorescent Protein (Book Chapter, Zacharias & Tsien) [zacharias]
All Medline abstracts: PubMed | HubMed

GFP as a measure of gene expression

  1. Leveau JH and Lindow SE. Predictive and interpretive simulation of green fluorescent protein expression in reporter bacteria. J Bacteriol. 2001 Dec;183(23):6752-62. DOI:10.1128/JB.183.23.6752-6762.2001 | PubMed ID:11698362 | HubMed [Lindow]
  2. Iafolla MA, Mazumder M, Sardana V, Velauthapillai T, Pannu K, and McMillen DR. Dark proteins: effect of inclusion body formation on quantification of protein expression. Proteins. 2008 Sep;72(4):1233-42. DOI:10.1002/prot.22024 | PubMed ID:18350571 | HubMed [Iafolla]
  3. Bagh S, Mazumder M, Velauthapillai T, Sardana V, Dong GQ, Movva AB, Lim LH, and McMillen DR. Plasmid-borne prokaryotic gene expression: sources of variability and quantitative system characterization. Phys Rev E Stat Nonlin Soft Matter Phys. 2008 Feb;77(2 Pt 1):021919. DOI:10.1103/PhysRevE.77.021919 | PubMed ID:18352063 | HubMed [Bagh]
  4. Dong GQ and McMillen DR. Effects of protein maturation on the noise in gene expression. Phys Rev E Stat Nonlin Soft Matter Phys. 2008 Feb;77(2 Pt 1):021908. DOI:10.1103/PhysRevE.77.021908 | PubMed ID:18352052 | HubMed [Dong]
All Medline abstracts: PubMed | HubMed