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. Copyright (C) 1998 Elsevier Science B.V.
Bibliographical noteFunding Information:
We would like to thank R. Tsien, W.P.C. Stemmer and S. Falkow for kindly providing Gfp-encoding genes. We also thank D. Bernlohr, J.W. Bodley and P. Hieter for providing the strains and plasmids used in this study. We would like to thank Mark Sanders and David Gartner of the Imaging Center, University of Minnesota, and Anurag Khetan of the Department of Chemical Engineering and Materials Science, University of Minnesota, for their expert assistance in acquisition and analysis of confocal microscopic images. This work was supported in part by the National Science Foundation (BES-9414385).
- Chinese hamster ovary
- Green fluorescent protein
- Saccharomyces cerevisiae