Difference between revisions of "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==
 
+
* From jellyfish ''Aeqorea victoria''
The fluorophore is tyrosine Y66 and the surrounding amino acids of Ser(65)-Tyr(66)-Gly(67) are also critical <cite>li97</cite>.
+
*[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=1169893 wtGFP sequence] on NCBI
 +
* 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>
 +
 +
==Mutations==
 +
* R96A: ''slows'' cyclization reaction from minutes to months
 +
* 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
 +
#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>
 
[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 12:10, 27 September 2006

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. FACS-optimized mutants of the green fluorescent protein (GFP). Gene. 1996;173(1 Spec No):33-8. PubMed ID:8707053 | HubMed [Cormack]
  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. 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]
All Medline abstracts: PubMed | HubMed