Abstract
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 NFkappaB binding sequences and monitoring tumor necrosis factor alpha-mediated NFkappaB 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 NFkappaB translocation to the nucleus.
Highlights
Because of its detected green fluorescence, the green fluorescent protein (GFP)1 from the jellyfish Aequorea victoria is a widely used reporter in studies of gene expression and protein localization [1,2,3,4]
To determine whether the PEST domain can induce Enhanced GFP (EGFP) degradation, we appended it to the Destabilized EGFP as a Transcription Reporter
We destabilized EGFP by appending the degradation domain of mouse ornithine decarboxylase (MODC) to the C terminus of EGFP
Summary
Because of its detected green fluorescence, the green fluorescent protein (GFP)1 from the jellyfish Aequorea victoria is a widely used reporter in studies of gene expression and protein localization [1,2,3,4]. The degradation domain of MODC dramatically decreased the half-life of EGFP in mammalian cells to 2 h. We mutated key amino acids in the PEST sequence of the fusion protein and identified several mutants with different half-lives.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have