COMPARISON OF MUTANT FORMS OF THE GREEN FLUORESCENT PROTEIN AS EXPRESSION MARKERS IN CHINESE-HAMSTER OVARY (CHO) AND SACCHAROMYCES-CEREVISIAE CELLS

Several green fluorescent protein (Gfp) mutants with increased cellula r fluorescence compared to the wildtype protein have recently been gen erated. We have expressed and compared wildtype Gfp and mutants S65T, F100S/ M154T/V164A, F64L/S65T, and S65A/V68L/S72A under identical grow th conditions in CHO and Saccharomyces cerevisiae cells. The results s uggest that the last two Gfp mutants are the best candidates as report er proteins, and they provide a high signal-to-noise ratio in both sys tems. Single gene copy expression of these mutant forms is easily dete ctable over background autofluorescence. All Gfps are highly stable wi thin cells, with an estimated 1/2-life between 7 h (wildtype) and 70 h (F100S/M154T/V164A) in S. cerevisiae cells. Although this limits thei r use in examining rapid cellular events without further modification, Gfp is expected to be a useful marker for monitoring the physiologica l state of cells in bioreactors using on-line probes. (C) 1998 Elsevie r Science B.V. All rights reserved.