GFP

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(GFP Variants)
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*[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=1169893 wtGFP on NCBI]
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==General Info==
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* From jellyfish ''Aeqorea victoria''
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The fluorophore is tyrosine Y66 and the surrounding amino acids of Ser(65)-Tyr(66)-Gly(67) are also critical <cite>li97</cite>.
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*[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=1169893 wtGFP sequence] on NCBI
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* 238 amino acids
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* The fluorophore is tyrosine Y66 and the surrounding amino acids of Ser(65)-Tyr(66)-Gly(67) are also critical <cite>zacharias</cite>.
==GFP Variants==
==GFP Variants==
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*[[Emerald]]
*[[Emerald]]
* EGFP F64L, S65T + optimized for human codons with 35-fold increase in fluorescence over GFP <cite>li97</cite>
* EGFP F64L, S65T + optimized for human codons with 35-fold increase in fluorescence over GFP <cite>li97</cite>
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==Mutations==
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* R96A: ''slows'' cyclization reaction from minutes to months
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* S65T: 5-6x increase in amplitude and red shift
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* Y203I: eliminate excitation peak at 475nm, leaving lower peak of 399nm. emission remains at 511nm, producing large Stokes shift
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* S65(G|T) and T203(Y|F|W|H) -> YFP
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* Y66W: GFP -> CFP
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* Y66H: GFP -> BFP (more blue than CFP), dim, easily photobleached
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* Y66F: excitation 360nm, emission 442nm
==References==
==References==
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#Cormack pmid=8707053
#Cormack pmid=8707053
#Andersen pmid=9603842
#Andersen pmid=9603842
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#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)]
</biblio>
</biblio>
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[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)]
 

Revision as of 15:10, 27 September 2006

Contents

General Info

  • From jellyfish Aeqorea victoria
  • wtGFP sequence on NCBI
  • 238 amino acids
  • The fluorophore is tyrosine Y66 and the surrounding amino acids of Ser(65)-Tyr(66)-Gly(67) are also critical [1].

GFP Variants

  • GFPmut3b - S65G, S72A
  • GFPmut3* - mutations from wt: S2R,S65G,S72A
    • This is GFPmut3b[2] with a degredation tag attached + an accidental mutation at position 2 that doesn't affect function according to the authors.[3]
  • Emerald
  • EGFP F64L, S65T + optimized for human codons with 35-fold increase in fluorescence over GFP [4]

Mutations

  • R96A: slows cyclization reaction from minutes to months
  • S65T: 5-6x increase in amplitude and red shift
  • Y203I: eliminate excitation peak at 475nm, leaving lower peak of 399nm. emission remains at 511nm, producing large Stokes shift
  • S65(G|T) and T203(Y|F|W|H) -> YFP
  • Y66W: GFP -> CFP
  • Y66H: GFP -> BFP (more blue than CFP), dim, easily photobleached
  • Y66F: excitation 360nm, emission 442nm

References

  1. Molecular Biology and Muation of Green Fluorescent Protein (Book Chapter, Zacharias & Tsien) [zacharias]
  2. Cormack BP, Valdivia RH, and Falkow S. . pmid:8707053. PubMed HubMed [Cormack]
  3. Li X, Zhang G, Ngo N, Zhao X, Kain SR, and Huang CC. . pmid:9353317. PubMed HubMed [li97]
  4. Andersen JB, Sternberg C, Poulsen LK, Bjorn SP, Givskov M, and Molin S. . pmid:9603842. PubMed HubMed [Andersen]
All Medline abstracts: PubMed HubMed
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