Generation of Destabilized Green Fluorescent Protein as a Transcription Reporter

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 NFB 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.Because of its easily 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 (1-4). GFP fluorescence does not require any substrate or cofactor (5); hence it is possible to use it in many species for live cell detection purposes. The fluorescence of GFPs is dependent on the key sequence Ser-Tyr-Gly (amino acids 65-67). This sequence undergoes spontaneous oxidation to form a cyclized chromophore (6). Enhanced GFP (EGFP) contains mutations of Ser to Thr at amino acid 65 and Phe to Leu at position 64 and is encoded by a gene with humanoptimized codons (7-9). Crystallographic structures of wildtype GFP and the mutant S65T reveal that the GFP tertiary structure resembles a barrel (10, 11). GFP is a single chain polypeptide of 238 amino acids (12). Most of these amino acids form ␤ sheets that are compacted through an antiparallel structure to form the barrel. An ␣-helix containing the chromophore is located inside the barrel, which shields it from the external environment. The compact structure makes GFP very stable under a variety of conditions, including treatment with protease (1). The stability of GFP limits its application in some studies, including transcriptional induction studies.Cellular proteins differ widely in their stabilities. Rapid turnover in proteins is often caused by signals that induce protein degradation. In some cases, the signal is a primary sequence such as the PEST sequence, a sequence possibly correlated with protein degradation (13,14)...

Inactivation of the type I interferon pathway reveals long double‐stranded RNA‐mediated RNA interference in mammalian cells

“…Notably, introduction of dsRNA‐ GFP but not dsRNA‐ RL led to a reduction in GFP expression at 2 days post‐transfection (Fig 1C). To increase temporal resolution, we switched to mESCs expressing a destabilised form of GFP (d2GFP), which has a reduced half‐life (Li et al , 1998). At 6 h post‐transfection (h.p.t.…”

Physiological regulation of transgene expression by a lentiviral vector containing the A2UCOE linked to a myeloid promoter

“…Mobilized CD34 þ cells were transduced with the SEW, MEW and UrMEW constructs expressing the destabilized version of GFP (d2eGFP). 32 D2eGFP expression was monitored in vitro 3 days post-transduction in HSPCs (CD133 þ /CD34 þ ) or after granulocytic differentiation 1 week later (CD11b þ /CD15 þ /CD66c þ ) (Supplementary Figure 5). Consistent with the observations made in murine cells, the SFFV-containing vector mediated d2eGFP expression in both cell subsets analyzed, whereas the MRP8 promoter was inactive in CD133 þ /CD34 þ cells, but the signal intensity was also moderate Figure 6).…”

Mutant SOD1 detoxification mechanisms in intact single cells

“…26 This region contains a PEST amino-acid sequence that targets the protein for degradation and results in rapid protein turnover. ECFP-PEST has a half-life of approximately 2 h. 26 In our experiments, the ECFP-PEST vector was cotransfected with a conventional EYFP expression vector to allow normalization of ECFP-PEST fluorescence to concentration differences within (i.e., compartmentalization) and between (i.e., transfection efficiency) cells. The image intensity values were corrected for the laser power setting needed to obtain proper photon statistics under the various conditions.…”

“…In order to find evidence for altered proteasome function in intact single cells expressing mtSOD1, we utilized a destabilized ECFP containing a PEST sequence (ECFP-PEST) for degradation targeting and, consequently, rapid turnover (t 1/2 ¼ 72 h 26 ). Transfection of ECFP-PEST results in negligible steadystate levels and only faint ECFP fluorescence, unless proteasomal degradation is impaired 26 ; Figure 4a and b). A concentration reference was provided by coexpression of (non-destabilized) EYFP to standardize ECFP-PEST fluorescence against concentration differences.…”