화학공학소재연구정보센터
Biochemical and Biophysical Research Communications, Vol.330, No.2, 454-460, 2005
Caspase-3 sensitive signaling in vivo in apoptotic HeLa cells by chemically engineered intramolecular fluorescence resonance energy transfer mutants of green fluorescent protein
Green fluorescent protein (UV5) was re-engineered to remove native cysteine residues, and a new cysteine was introduced near the C-terminus, similar to 20 angstrom from the native fluorophore, for site-specific attachment of chemical fluorophores. The resultant efficient intramolecular FRET quenched GFP emission and gave a new emission band from the conjugated fluorophore. Caspase-3 cleavage of constructs with a caspase-3 sequence near the C-terminus in the sequence between the native fluorophore and the new cysteine, located C-terminal to the caspase site, destroyed the FRET, the emitted color reverting to that of unmodified GFP. This process was demonstrated in vitro with caspase-3 and lysates from cells undergoing apoptosis. Real-time emission changes for the Alexa Fluor 532 conjugate of this GFP, studied quantitatively in vivo for single HeLa cells using the ratios of fluorescence at the red and green maxima by confocal microscopy, showed that caspase-3 action in the cytosol preceded that in the nucleus. (c) 2005 Elsevier Inc. All rights reserved.