Nuclear localization of enhanced green fluorescent protein homomultimers

The green fluorescent protein (GFP) and its variants are used in many studies to determine the subcellular localization of other proteins by analyzing fusion proteins. The main problem for nuclear localization studies is the fact that, to some extent, GFP translocates to the nucleus on its own. Because the nuclear import could be due to unspecific diffusion of the relatively small GFP through the nuclear pores, we analyzed the localization of multimers of a GFP variant, the enhanced GFP (EGFP). By detecting the fluorescence of the expressed proteins in gels after nonreducing SDS-PAGE, we demonstrate the integrity of the expressed proteins. Nevertheless, even EGFP homotetramers and homohexamers are found in the nuclei of the five analyzed mammalian cell lines. The use of fusion constructs of small proteins with multimeric EGFP alone, therefore, is not adequate to prove nuclear import processes. Fusion to tetrameric EGFP in combination with a careful quantification of the fluorescence intensities in the nucleus and cytoplasm might be sufficient in many cases to identify a significant difference between the fusion protein and tetrameric EGFP alone to deduce a nuclear localization signal.     

A desolvation model for trifluoroethanol-induced aggregation of enhanced green fluorescent protein

Studies of amyloid disease-associated proteins in aqueous solutions containing 2,2,2-trifluoroethanol (TFE) have shown that the formation of structural intermediates is often correlated with enhanced protein aggregation. Here, enhanced green fluorescent protein (EGFP) is used as a model protein system to investigate the causal relationship between TFE-induced structural transitions and aggregation. Using circular dichroism spectroscopy, light scattering measurements, and transmission electron microscopy imaging, we demonstrate that population of a partially α-helical, monomeric intermediate is roughly correlated with the growth of β-sheet-rich, flexible fibrils for acid-denatured EGFP. By fitting our circular dichroism data to a model in which TFE-water mixtures are assumed to be ideal solutions, we show that increasing entropic costs of protein solvation in TFE-water mixtures may both cause the population of the intermediate state and increase aggregate production. Tertiary structure and electrostatic repulsion also impede aggregation. We conclude that initiation of EGFP aggregation in TFE likely involves overcoming of multiple protective factors, rather than stabilization of aggregation-prone structural elements.     

Oviduct-specific enhanced green fluorescent protein expression in transgenic chickens

In this study, we confirmed the ability of the 2-kb promoter fragment of the chicken ovalbumin gene to drive tissue-specific expression of a foreign EGFP gene in chickens. Recombinant lentiviruses containing the EGFP gene were injected into the subgerminal cavity of 539 freshly laid embryos (stage X). Subsequently the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. Twenty-four chicks (G0) were hatched and screened for EGFP with PCR. Two chicks were identified as transgenic birds (G1), and these founders were mated with wild-type chickens to generate transgenic progeny. In the generated transgenic hens (G2), EGFP was expressed specifically in the tubular gland of the oviduct. These results show the potential of the chicken ovalbumin promoter for the production of biologically active proteins in egg white.     

A conditional mouse line for lineage tracing of Sox9 loss-of-function cells using enhanced green fluorescent protein

Traditionally, conditional knockout studies in mouse have utilized the Cre or Flpe technology to activate the expression of reporter genes such as lacZ or PLAP. Employing these reporter genes, however, requires tissue fixation. To make way for downstream in vivo or in vitro applications, we have inserted enhanced green fluorescent protein (EGFP) into the endogenous Sox9 locus and generated a novel conditional Sox9 null allele, by flanking the entire Sox9 coding region with loxP sites and inserting an EGFP reporter gene into the 3′-UTR allowing for EGFP to be expressed upon Sox9 loss of function yet under the control of the endogenous Sox9 promoter. Mating this new allele to any Cre-expressing line, the fate of Sox9 null cells can be traced in the cell type of interest in vivo or in vitro after fluorescence-activated cell sorting.     

Improved technique for detection of enhanced green fluorescent protein in transgenic mice.

One of the most exciting recent advances in cell biology is the possibility to use the green fluorescent protein and its various mutated forms as reporter proteins in studies carried out in vitro and in vivo. In the present study, several detection techniques for the enhanced green fluorescent protein (EGFP) were compared in transgenic mice, using fluorescence and confocal microscopy. In addition, different tissue preparation techniques (squash preparations, vibratome sections, frozen sections) were evaluated. As a model we used transgenic mice expressing EGFP under the control of a 5.0-kb fragment of the glutathione peroxidase isoenzyme 5 protein promoter (GPX5-EGFP) or under a 3.8-kb fragment of the cysteine rich protein-1 promoter (CRISP1-EGFP). In the GPX5-EGFP mice, expression of EGFP was observed in the distal part of the caput epididymis, while the CRISP1 promoter directed EGFP expression in the tubular compartment of the testis. Among the various tissue preparation procedures tested, the best morphological and histological preservation, and reproducibility in EGFP detection, were obtained using frozen sections after a slow tissue-freezing protocol developed in the present study. After slow tissue freezing, specimens of testis and epididymis could be stored at -70 degrees C for at least six weeks without any affect on EGFP fluorescence. Hence, the method developed offers the possibility to analyze EGFP fluorescence in tissues several weeks after specimen collection. The sensitivity achieved was equal to that found in immunohistochemistry, applying biotin-streptavidin-FITC detection. Confocal microscopy is known to have the advantage that fluorescence can be detected from cells in different layers. This was found to be important as regards detecting EGFP fluorescence because the fluorescence was destroyed at the cut surfaces of sections produced by either vibratome or cryomicrotome.     

Imidazole as a catalyst for in vitro refolding of enhanced green fluorescent protein

Imidazole is a reagent widely used in protein purifying processes. Here, we reveal a novel chaperone-like activity for imidazole using enhanced green fluorescent protein (EGFP) as a model protein. Experimental results showed that imidazole acted as an effective catalyst for refolding of the chemically denatured EGFP and suppressor for the heat-induced aggregation of EGFP. The refolding kinetics was determined in real time. Both the recovering yield and refolding rate of denatured EGFP in the presence of imidazole were increased. The studies on elucidating the mechanism show that imidazole may catalyze the prolyl cis/trans isomerization and the possible mechanism was discussed. To our knowledge, there are no data on the effect of imidazole on protein folding. Considering the prolyl isomerization is the rate-limited step for refolding of most proteins and aggregation is a universal serious problem for biotechnology, imidazole thus represents a previous unknown type of protein-folding catalyst.     

Use of enhanced green fluorescent protein to determine pepsin at high sensitivity

A fluorometric assay for pepsin and pepsinogen was developed using enhanced green fluorescent protein (EGFP) as a substrate. Acid denaturation of EGFP resulted in a complete loss of fluorescence that was completely reversible on neutralization. In the proteolytic assay procedure, acid-denatured EGFP was digested by pepsin or activated pepsinogen. After neutralization, the remaining amount of undigested EGFP refolded and was determined by fluorescence. Under standard digestion conditions, 4.8-24.0 ng pepsin or pepsinogen was used. Using porcine pepsin as a standard, 38+/-6.7 ng EGFP was digested per min-1 ng pepsin-1. Activated porcine pepsinogen revealed a similar digestion rate (37.2+/-5.2 ng EGFP min-1 ng activated pepsinogen-1). The sensitivity of the proteolysis assay depended on the time of digestion and the temperature. Increasing temperature and incubation time allowed quantification of pepsin or pepsinogen in a sample even in the picogram range. The pepsin-catalyzed EGFP digestion showed typical Michaelis-Menten kinetics. Km and Vmax values were determined for the pepsin and activated pepsinogen. Digestion of EGFP by pepsin revealed distinct cleavage sites, as analyzed by SDS-PAGE.     

Construction and characterization of a fluorescent sendai virus carrying the gene for envelope fusion protein fused with enhanced green fluorescent protein

Sendai virus (SeV) is an enveloped virus with a non-segmented negative-strand RNA genome. SeV envelope fusion (F) glycoproteins play crucial roles in the viral life cycle in processes such as viral binding, assembly, and budding. In this study, we developed a viable recombinant SeV designated F-EGFP SeV/ΔF, in which the F protein was replaced by an F protein fused to EGFP at the carboxyl terminus. Living infected cells of the recombinant virus were directly visualized by green fluorescence. The addition of EGFP to the F protein maintained the activities of the F protein in terms of intracellular transport to the plasma membrane via the ER and the Golgi apparatus and fusion activity in the infected cells. These results suggest that this fluorescent SeV is a useful tool for studying the viral binding, assembly, and budding mechanisms of F proteins and the SeV life cycle in living infected cells.     

Attenuation of green fluorescent protein half-life in mammalian cells

The half-life of the green fluorescent protein (GFP) was determined biochemically in cultured mouse LA-9 cells. The wild-type protein was found to be stable with a half-life of approximately 26 h, but could be destabilized by the addition of putative proteolytic signal sequences derived from proteins with shorter half-lives. A C-terminal fusion of a PEST sequence from the mouse ornithine decarboxylase gene reduced the half-life to 9.8 h, resulting in a GFP variant suitable for the study of dynamic cellular processes. In an N-terminal fusion containing the mouse cyclin B1 destruction box, it was reduced to 5.8 h, with most degradation taking place at metaphase. The combination of both sequences produced a similar GFP half-life of 5.5 h. Thus, the stability of this marker protein can be controlled in predetermined ways by addition of the appropriate proteolytic signals.     

Reversible photobleaching of enhanced green fluorescent proteins

Color variants of green fluorescent protein (GFP) are increasingly used for multicolor imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching (FRAP). Here we show that experimental settings commonly used in these imaging experiments may induce an as yet uncharacterized reversible photobleaching of fluorescent proteins, which is more pronounced at acidic pH. Whereas the reversible photobleaching spectrum of eCFP corresponds to its absorption spectrum, reversible photobleaching spectra of yellow variants resemble absorption spectra of their protonated states. Fluorescence intensities recover spontaneously with time constants of 25-58 s. The recovery of eCFP can be further accelerated by illumination. The resulting steady-state fluorescence reflects a variable equilibrium between reversible photobleaching, spontaneous recovery, and light-induced recovery. These processes can cause significant artifacts in commonly applied imaging techniques, photobleach-based FRET determinations, and FRAP assays.     

Development of an assay for beta-lactam hydrolysis using the pH-dependence of enhanced green fluorescent protein

An assay has been developed utilizing the pH-dependent fluorescence of enhanced green fluorescent protein (EGFP). This photoprotein allows for the study of kinetic properties of hydrolytic enzymes based on the production of protons. As a model system, beta-lactamase, a well-characterized enzyme responsible for antibiotic resistance in many bacteria, was used. More specifically, EGFP and beta-lactamase were genetically fused using overlap extension PCR and incorporated into a bacterial expression vector. The vector was subsequently transformed into Escherichia coli, and the fusion protein was expressed and purified. beta-Lactamase catalyzes the hydrolysis of the beta-lactam ring of ampicillin. This causes a decrease in the local pH, which in turn changes the spectral properties of EGFP. This property was utilized to perform enzyme kinetic studies on the new fusion protein as well as on the beta-lactamase inhibitor, sulbactam. The assay can be used to evaluate substrates and inhibitors of beta-lactamase in a format that should be amenable to high-throughput screening.     

Preparation of heteroduplex enhanced green fluorescent protein plasmid for in vivo mismatch repair activity assay

Preparation of heteroduplexes in large quantities with high purity is essential for the measurement of DNA mismatch repair (MMR) activity. Here we report a rapid, less labor-intensive method for the preparation of a heteroduplex plasmid that expresses the enhanced green fluorescent protein (EGFP) if the mismatch is repaired correctly. The method involves the use of a wild-type and a mutated EGFP expression plasmid and a few steps of enzymatic digestion. When the constructed heteroduplex EGFP plasmid was transfected into MMR-proficient and -deficient cell lines, the number of EGFP-expressing cells was much higher in the MMR-proficient cells than in the MMR-deficient cells, suggesting that the heteroduplex can be used for MMR activity assay in live model systems.     

pGreen-S: A clone vector bearing absence of enhanced green fluorescent protein for screening recombinants

The bacterial cloning vector, pGreen-S, was constructed by inserting the enhanced green fluorescent protein (EGFP) gene at the XbaI restriction site of pUC18 plasmid. When expressed in Escherichia coli DH5α produced colonies that were an absinthe green color under daylight and strongly fluorescent green under longwave ultraviolet light. The pGreen-S vector was used to select for directional insert based on the loss of green fluorescence in recombinant colonies that was caused by the absence of EGFP. The EGFP reporter system differs from the conventional complementation of lacZ, making screening recombinants simpler, less expensive, and more effective.     

Preparation of a bio-immunoreagent between ZZ affibody and enhanced green fluorescent protein for immunofluorescence applications

In the present study, we constructed plasmid pUC-ZZ-EGFP to express Pro-ZZ-EGFP using ZZ peptide (a synthetic artificial IgG-Fc-fragment-binding protein derived from the B domain of staphylococcal protein A) and enhanced green fluorescent protein (EGFP). Without induction with isopropyl-β-d-thiogalactopyranoside, the chimeric protein was effectively expressed in Escherichia coli HB101. Its affinity constant binding IgG was 2.6 × 10⁸ M⁻¹ obtained by competitive enzyme-linked immunosorbent assay, indicating that the ZZ peptide retains the native structure in Pro-ZZ-EGFP. The application of immunofluorescence assay for detecting the Mycoplasma pneumoniae IgG antibody, Pro-ZZ-EGFP, exhibited a good signal comparable in brightness and fluorescence pattern with the signal generated using the fluorescein isothiocyanate-labeled anti-human IgG. The result indicates that Pro-ZZ-EGFP possesses great potential for clinical immunofluorescence IgG test as an alternative versatile fluorescent antibody.     

Impact of embryonic expression of enhanced green fluorescent protein on early mouse development

The impact of embryonic enhanced green fluorescent protein (EGFP)-expression on development is not clear. In this study, we comprehensively assessed EGFP-expression pattern and its effect on early mouse development, following pronuclear-microinjection of the EGFP-transgene, containing chicken-beta-actin promoter and cytomegalovirus enhancer. Preimplantation embryos exhibited differential EGFP-expression patterns. While blastocyst development of non-expressing embryos was 77.3+/-1.8%, that of expressing embryos was only 43.9+/-1.6% (P<0.0001). Developmental competence of embryos negatively correlated (r=-0.99) with the levels of EGFP-expression. Faint-, moderate-, and intense-expressing embryos developed to 83.1+/-5.3%, 50+/-5%, and 9.5+/-3.9% blastocysts, respectively (P<0.002). Interestingly, blastocysts expressing faint-moderate levels of EGFP were developmentally competent through the post-implantation period and delivered viable transgenic 'green' mice, following embryo transfer. These results indicate that hyper-expression of EGFP affects preimplantation development and faint-moderate level of its expression is compatible with normal embryogenesis in the mouse.     

Transient immunosuppression stops rejection of virus-transduced enhanced green fluorescent protein in rabbit retina

The expression of lentivirus-transduced enhanced green fluorescent protein (EGFP) was detectable in rabbit retinal pigment epithelium (RPE) within 3 to 5 days after subretinal injection of the vector. Within 2 to 3 weeks, EGFP-expressing cells were eliminated by rejection. In the current experiments, we monitor serum antibody titers for EGFP before and after transduction and determine whether systemic immunosuppression prevents recognition of EGFP by the immune system. While all control rabbits developed antibodies against EFGP and showed signs of rejection, no such evidence was observed with animals which received immunosuppression. One month of systemic immunosuppression permanently prevented rejection of RPE with EGFP expression. Fluorescence has been maintained for more than a year. If a control eye was injected with the same virus after terminating immunosuppression, both eyes showed signs of rejection. The lack of rejection is not due to tolerance but to a failure of the animals to detect the foreign protein. Detection must depend upon a brief window of time after surgery needed to introduce the vector, perhaps related to a concurrent but transient inflammation. This strategy may be useful in managing other types of rejection in the retina.     

Novel synthetic signal peptides for the periplasmic secretion of green fluorescent protein in Escherichia coli

New secretion vectors containing synthetic signal peptides were constructed to study the periplasmic translocation of green fluorescent protein (GFP) in Escherichia coli. These constructs encode synthetic signal peptides spA and spD fused to the amino terminal end of GFP, and expressed from T7/lac promoter in the BL21DE3 strain by induction with IPTG. The recombinant protein was detected in both the cytoplasmic and periplasmic fractions. Fluorescence analysis revealed that recombinant proteins with signal peptides were not fluorescent, indicating translocation to periplasmic space. In contrast, recombinant proteins without signal peptide were fluorescent. These results indicate that the expressed recombinant proteins were translocated into the periplasm. Therefore, the synthetic signal peptides derived from signal peptides of Bacillus sp. could efficiently secrete the heterologous proteins to the periplasmic space of E. coli.     

Isolation of peroxisome-defective CHO mutant cells using green fluorescent protein

The authors constructed a recombinant green fluorescent protein (GFP) (PTS-GFP), which carries peroxisome targeting signal (PTS1 or PTS2) as an additional sequence, by polymerase chain reaction. The gene encoding for the recombinant GFP was constructed into an eukaryotic expression vector, and stable transformant of CHO cell expressing PTS-GFP was isolated, following the transfection of the plasmid encoding for the GFP. Each expressed PTS-GFP appeared to be localized in peroxisomes, because the GFP was observed in cellular structures, as was catalase. The observation proposed a visual screening procedure for isolating peroxisome-defective mutant. Following an enrichment of mutant cells by use of 9-(1′-pyrene)nonanol/ultraviolet irradiation (P9OH/UV) method, five peroxisome-defective mutants were isolated by pursuing the fluorescent signals from GFP. Two mutants (SK24 and SK32) were isolated from CHO cells expressing PTS1-GFP, and three mutants (PT13, PT32, and PT54) were isolated from cells expressing PTS2-GFP. Four mutants, except for PT13, showed cytosolic distributions of both PTS-GFP and catalase. On the other hand, mutant PT13 showed a cytosolic distribution on PTS2-GFP, but a peroxisomal distribution on catalase. Cell fusion analysis between SK24 mutant and other mutants indicated that PT54 mutant is in the same complementation group (CG) as SK24, but that SK32, PT13, and PT32 mutants are in different complementation group(s) from SK24.