I'm using GFP as a quantitative marker to on-line and flow cytometrically
track protein expression in Saccharomyces cerevisiae. Since, due to its
folding and autooxidation, at least 2.5 h (wt GFP in centromeric plasmid)
and even up to 4.5 h (wt GFP in 100-copy plasmid) are needed to obtain final
fluorescence, wt GFP turned out to be useless in this respect. Also GFPS65T
remains suboptimal since, when even cloned in a centromeric plasmid, still
45 min to 1 h are needed. Fixing the cells and vortexing them to stimulate
autooxdation does not help. In fact, the fluorescence most of the time
completely disappears probably due to a stimulated entering of the fixing
agent inside the cells (e.g also when using EtOH70%, contrary to many
literature reports,...). Besides we'd like to track GFP expression without
disturbing the cells (e.g. by fixation). Are there some new and faster GFPs
around of which I do not know ? Any suggestions on how to stimulate the
folding/autoxidation in vivo without disturbing the cells ?
Many thanks,
Peter De wulf
