A highly sensitive assay for proteases using staphylococcal protein A fused with enhanced green fluorescent protein

Enhanced green fluorescent protein (EGFP) was fused with staphylococcal protein A (SpA) and used as a substrate for proteases. An SpA-EGFP assay was done in three steps: (i) digestion of SpA-EGFP by proteases, (ii) addition of rabbit IgG immobilized on Sepharose beads, and (iii) measurement of the fluorescence intensity of supernatant. The assay was sensitive enough to measure picogram levels of trypsin and chymotrypsin, and may be applicable to various other proteases as one of the most sensitive methods.     

Improved method to raise polyclonal antibody using enhanced green fluorescent protein transgenic mice

Recombinant fusion protein is widely used as an antigen to raise antibodies against the epitope of a target protein. However, the concomitant anticarrier antibody in resulting antiserum reduces the production of the desired antibody and brings about unwanted non-specific immune reactions. It is proposed that the carrier protein transgeulc animal could be used to solve this problem. To validate this hypothesis, enhanced green fluorescent protein （EGFP） transgenic mice were produced. By immunizing the mice with fusion protein His6HAtag-EGFP, we showed that the antiserum from the transgenic mice had higher titer antibody against His6HA tag and lower titer antibody against EGFP compared with that from wild-type mice. Therefore, this report describes an improved method to raise high titer antipeptide polyclonal antibody using EGFP transgenic mice that could have application potential in antibody preparation.     

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.     

Viral RNA-based transfection and expression of enhanced green fluorescent protein in the parasitic protozoan Eimeria stiedae

Eimeria stiedae(E.stiedae),the most common protozoan pathogen of rabbits,causes coccidiosis.This disease is problematic for rabbit breeders [1].There is little information about the cellular and molecular biology of this pathogen and a lack of genetic tools.Genetic manipulation is necessary to advance our understanding of parasite biology and ultimately develop vaccines and treat diseases.DNA-mediated and viral RNA-mediated transfection systems have successfully been established in many protozoan parasites [2,3].It was discovered that E.stiedae possesses a dsRNA virus(EsRV1),and more recently,the genome of this virus has been sequenced.To date,however,there is no report on the use of viral RNA-mediated transfection for E.stiedae.In this study,we report a transient viral RNA-mediated transfection of E.stiedae using enhanced green fluorescent protein(EGFP)as a reporter gene.Our study provides a valuable genetic tool for studying the biology of E.stiedae.     

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.     

Development of a method for screening short-lived proteins using green fluorescent protein

We have developed a screening technology for the identification of short-lived proteins. A green fluorescent protein (GFP)-fusion cDNA library was generated for monitoring degradation kinetics. Cells expressing a subset of the GFP-cDNA expression library were screened to recover those in which the fluorescence signal diminished rapidly when protein synthesis was inhibited. Thirty clones that met the screening criteria were characterized individually. Twenty-three (73%) proved to have a half-life of 4 hours or less.     

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.     

Quantitation of green fluorescent protein using a gel-based imaging method

Green fluorescent protein (GFP) is a versatile reporter protein and has been widely used in biological research. However, its quantitation requires expensive equipment such as a spectrofluorometer. In the current study, a gel documentation imaging system using a native polyacrylamide gel for the quantitation of GFP was developed. The assay was evaluated for its precision, linearity, reproducibility, and sensitivity in the presence of Escherichia coli cells and was compared with the spectrofluorometric method. Using this newly established, gel-based imaging technique; the amount of GFP can be quantified accurately.     

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.     

Modification of enhanced green fluorescent protein for secretion out of cells

Since its discovery approximately 20 years ago, green fluorescent protein (GFP) has become one of the most widely used reporter proteins. GFP has been used in a variety of living organisms, ranging from E. coli to higher eukaryotes, such as plants and animals. The biggest advantage of using this reporter protein is that it can be used to monitor in vitro and in vivo gene expression. One important limitation, however, is its inability to be secreted out of cells. For this reason, it has been difficult to directly measure the expression level of the regulatory sequence of a gene of interest quantitatively. To overcome this drawback, we have modified the enhanced green fluorescent protein gene (EGFP), a derivative of GFP, by adding a signal peptide sequence that encodes a rat follicle-stimulating hormone (FSH) β-subunit upstream of EGFP. Following the expression of this modified gene in several cell types, we have found efficient secretion of EGFP. Consequently, with the secreted protein, we could easily quantify the gene expression level with high reliability. Therefore, the use of our modified EGFP expression cassette would greatly facilitate the evaluation of regulatory sequences, such as promoters and enhancers. Further, it will also be very helpful in the study of transgenic livestock intended to use as bioreactors for mass production of pharmaceuticals.     

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 new method using enhanced green fluorescent protein (EGFP) to determine grazing rate on live bacterial cells by protists

A new method was developed for estimating the grazing rate of live bacteria by protists. Bacterial cells (Escherichia coli bearing plasmid pEGFP) expressing enhanced green fluorescent protein (EGFP) were used as a live bacterial tracer. Ciliates (Tetrahymena thermophila) were fed with EGFP-tagged bacterial cells, and the individual cells taken up by the ciliates were detected by epifluorescence microscopy. The EGFP fluorescence was stable during the storage of samples fixed with glutaraldehyde. Comparison of clearance rates based on the uptake of EGFP-tagged live cells and fluorescently-labeled heat-killed cells suggested that the use of heat-killed cells underestimates the clearance rates. We suggest that EGFP-tagged bacteria are a useful tracer for determining protist bacterivory in culture and aquatic environments. Received: January 22, 2001 / Accepted: October 27, 2001     

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.     

A simple fluorescent method for quantitative determination of aortic protein uptake.

A quantitative system for direct protein tracing and measurement of net protein uptake in the aorta using fluorescein isothiocyanate conjugated bovine serum albumin (FITCBSA) is described. Using Wistar rats, a mean aortic FITCBSA net uptake of 29.7 times 10(-17) g FITCBSA per mum2 aortic endothelial surface area per 24 h was obtained. Intra-aortic localization of the FITCBSA was observed along the endothelium and the collagen-elastin bands. The values obtained using this FITCBSA system are comparable with those of a previously established isotopic technique measuring aortic albumin flux and reconfirm the previous findings of the existence of an albumin permeability gradient in the thoracic aorta.     

A high-throughput approach to promoter study using green fluorescent protein

Green fluorescent protein (GFP) is a reporter that has had a significant impact due to its many advantages over other reporter genes: it is autofluorescent, it enables in situ detection, it is relatively small, and it is also nontoxic. By cloning a gene promoter upstream of the gfp gene and exposing the living cells transformed with the fusion to the specific inducer or repressor, gene expression can be real-time monitored by continuous quantitative measurement of the green fluorescence emitted by GFP. In this work, a promoter study using promoter-gfp fusions was conducted in 96-well plates. Because they were placed in an automated incubating and shaking microplate reader, the wells functioned as microscale bioreactors, allowing for parallel experiments and data analysis. In the study described here, an overexpression promoter (pBAD promoter) and two comparatively weak promoters (sodA and acnA in Escherichia coli SoxRS regulon) were studied in both endpoint and kinetics formats. Our results with the pBAD promoter revealed insight on its regulation, which is tightly controlled by levels of arabinose and glucose. Results on weak oxidative stress promoters (for sodA and acnA genes) were striking in that significant induction was observed when they were under a superoxide stress in plates. They both displayed dose-dependent induction to paraquat-generated superoxide anion, with sodA leading acnA in strength and time. These results, spanning highly inducible promoters for protein overexpression and weakly inducible promoters of metabolic interest, demonstrate that the approach is relatively easily executed and can be used for quantitative and temporal promoter studies in a high throughput format.     

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.     

Concentrating rotavirus and recombinant enhanced green fluorescent protein fromE. coli using a temperature-sensitive hydrogel

Recombinant enhanced green fluorescent protein (EGFP) fromE. coli was concentrated 1.9 times by ultrafiltration using a temperature-sensitive hydrogel. Protein recovery and separation efficiency were 64% and 45%, respectively. Increased concentration of recombinant EGFP was confirmed by SDS-PAGE. Rotavirus was concentrated 3.2 times by ultrafiltration using a temperature-sensitive hydrogel, at 95% of virus recovery and 93% of separation efficiency. Hydrogel ultrafiltration appears to be an attractive alternative for the concentration of rotavirus and recombinant proteins fromE. coli.