Comparison of mutant forms of the green fluorescent protein as expression markers in Chinese hamster ovary (CHO) and Saccharomyces cerevisiae cells

Abstract

Several green fluorescent protein (Gfp) mutants with increased cellular fluorescence compared to the wildtype protein have recently been generated. We have expressed and compared wildtype Gfp and mutants S65T, F100S/ M154T/V164A, F64L/S65T, and S65A/V68L/S72A under identical growth conditions in CHO and Saccharomyces cerevisiae cells. The results suggest that the last two Gfp mutants are the best candidates as reporter proteins, and they provide a high signal-to-noise ratio in both systems. Single gene copy expression of these mutant forms is easily detectable over background autofluorescence. All Gfps are highly stable within cells, with an estimated 1/2-life between 7 h (wildtype) and 70 h (F100S/M154T/V164A) in S. cerevisiae cells. Although this limits their use in examining rapid cellular events without further modification, Gfp is expected to be a useful marker for monitoring the physiological state of cells in bioreactors using on-line probes.