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J Biol Chem, Vol. 273, Issue 52, 34970-34975, December 25, 1998
From CLONTECH Laboratories, Inc.,
Palo Alto, California 94303
The green fluorescent protein (GFP) is a widely
used reporter in gene expression and protein localization studies. GFP
is a stable protein; this property allows its accumulation and easy detection in cells. However, this stability also limits its application in studies that require rapid reporter turnover. We created a destabilized GFP for use in such studies by fusing amino acids 422-461
of the degradation domain of mouse ornithine decarboxylase (MODC) to
the C-terminal end of an enhanced variant of GFP (EGFP). The fusion
protein, unlike EGFP, was unstable in the presence of cycloheximide and
had a fluorescence half-life of 2 h. Western blot analysis
indicated that the fluorescence decay of EGFP-MODC-(422-461) was
correlated with degradation of the fusion protein. We mutated key amino
acids in the PEST sequence of EGFP-MODC-(422-461) and identified
several mutants with variable half-lives. The suitability of
destabilized EGFP as a transcription reporter was tested by linking it
to NF
B binding sequences and monitoring tumor necrosis factor
-mediated NFB activation. We obtained time course induction and
dose response kinetics similar to secreted alkaline phosphatase obtained in transfected cells. This result did not occur when unmodified EGFP was used as the reporter. Because of its
autofluorescence, destabilized EGFP can be used to directly correlate
gene induction with biochemical change, such as NFB translocation to
the nucleus.
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