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 - 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

• 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
• A206K: make monomeric

GFP as a measure of gene expression

pmid=11698362

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 |