GFP

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].
• Oxygen is required for maturation of the fluorophore

GFP Variants

• GFPmut3b - S65G, S72A
• GFPmut3* - mutations from wt: S2R,S65G,S72A
  • This is GFPmut3b[2] with an accidental mutation at position 2 that doesn't affect function according to the authors.[3]
• Emerald - F64L, S65T, S72A, N149K, M153T, I167T
• EGFP - added GTG as second codon, F64L, S65T + optimized for human codons with 35-fold increase in fluorescence over GFP [4]
• Other variants and their mutations can be found in Shaner supplementary table 2 [5]

Mutations

• F64L: improve folding @37C
• S65T: 5-6x increase in amplitude and red 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
• 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
• A206K: make monomeric

GFP as a measure of gene expression

6. 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 |

References

1. Molecular Biology and Muation of Green Fluorescent Protein (Book Chapter, Zacharias & Tsien)

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. DOI:10.1016/0378-1119(95)00685-0 |
3. 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. DOI:10.1128/AEM.64.6.2240-2246.1998 |
4. 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. DOI:10.1074/jbc.272.45.28545 |
5. 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 |
7. Tsien RY. The green fluorescent protein. Annu Rev Biochem. 1998;67:509-44. DOI:10.1146/annurev.biochem.67.1.509 |
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. DOI:10.1016/0378-1119(92)90691-h |

   original cloning of GFP