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BMC Biotechnol. 2003 May 22;3:5. Epub 2003 May 22.

Efficiently folding and circularly permuted variants of the Sapphire mutant of GFP.

Author information

1
Max-Planck-Institut für Neurobiologie Am Klopferspitz 18a 82152 Martinsried, Germany. zapata@neuro.mpg.de

Abstract

BACKGROUND:

The green fluorescent protein (GFP) has been widely used in cell biology as a marker of gene expression, label of cellular structures, fusion tag or as a crucial constituent of genetically encoded biosensors. Mutagenesis of the wildtype gene has yielded a number of improved variants such as EGFP or colour variants suitable for fluorescence resonance energy transfer (FRET). However, folding of some of these mutants is still a problem when targeted to certain organelles or fused to other proteins.

RESULTS:

By directed rational mutagenesis, we have produced a new variant of the Sapphire mutant of GFP with improved folding properties that turns out to be especially beneficial when expressed within organelles or as a fusion tag. Its absorption spectrum is pH-stable and the pKa of its emission is 4.9, making it very resistant to pH perturbation inside cells.

CONCLUSION:

"T-Sapphire" and its circular permutations can be used as labels of proteins or cellular structures and as FRET donors in combination with red-fluorescent acceptor proteins such as DsRed, making it possible to completely separate donor and acceptor excitation and emission in intensity-based FRET experiments.

PMID:
12769828
PMCID:
PMC161811
DOI:
10.1186/1472-6750-3-5
[Indexed for MEDLINE]
Free PMC Article

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