GFP
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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
- 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
References
- 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 |
- 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 |
- 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 |
- 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 |