Fluorometric quantification of green fluorescent protein in tobacco leaf extracts

The main use of green fluorescent protein (GFP) is as a reporter system, where the existence of the protein is usually determined visually using fluorescent microscopy. Although fluorescence-based quantification of GFP is possible, background fluorescence in plants and in plant extracts was observed by our group. Another phenomenon we observed that makes quantification difficult is the increased level of GFP fluorescence in Nicotiana benthamiana leaf extracts, probably the result of dimerization of GFP molecules promoted by interaction with some component(s) of tobacco extracts. In the current work, the background fluorescence was minimized and the enhancement of GFP fluorescence in tobacco extracts was eliminated with the addition of urea to the measured solution so that a simple quantification assay for the GFP in the tobacco extracts could be established.     

Enhanced fluorescent properties of an OmpT site deleted mutant of Green Fluorescent Protein

Background  

The green fluorescent protein has revolutionized many areas of cell biology and biotechnology since it is widely used in determining gene expression and for localization of protein expression. Expression of recombinant GFP in E. coli K12 host from pBAD24M-GFP construct upon arabinose induction was significantly lower than that seen in E. coli B cells with higher expression at 30°C as compared to 37°C in E. coli K12 hosts. Since OmpT levels are higher at 37°C than at 30°C, it prompted us to modify the OmpT proteolytic sites of GFP and examine such an effect on GFP expression and fluorescence. Upon modification of one of the two putative OmpT cleavage sites of GFP, we observed several folds enhanced fluorescence of GFP as compared to unmodified GFPuv (Wild Type-WT). The western blot studies of the WT and the SDM II GFP mutant using anti-GFP antibody showed prominent degradation of GFP with negligible degradation in case of SDM II GFP mutant while no such degradation of GFP was seen for both the clones when expressed in BL21 cells. The SDM II GFP mutant also showed enhanced GFP fluorescence in other E. coli K12 OmpT hosts like E. coli JM109 and LE 392 in comparison to WT GFPuv. Inclusion of an OmpT inhibitor, like zinc with WT GFP lysate expressed from an E. coli K12 host was found to reduce degradation of GFP fluorescence by two fold.     

Quantitative GFP fluorescence as an indicator of recombinant protein synthesis in transgenic plants

The utility of green fluorescent protein (GFP) for biological research is evident. A fluorescence-based method was developed to quantify GFP levels in transgenic plants and protein extracts. Fluorescence intensity was linear with increasing levels of GFP over a range that encompasses transgene expression in plants by the cauliflower mosaic virus 35S promoter. Standard curves were used to estimate GFP concentration in planta and in protein extracts. These values were consistent with ELISA measurements of GFP in protein extracts from transgenic plants, indicating that the technique is a reliable measure of recombinant GFP expression. The levels of in planta GFP expression in both homozygous and hemizygous plants was then estimated. Homozygous transgenic plants expressed twice the amount of GFP than hemizygous plants, suggesting additive transgene expression. This methodology may be useful to simplify the characterization of transgene expression in plants.Abbreviations ELISA Enzyme-linked immunosorbent assay - HRP Horseradish peroxidase - GFP Green fluorescent protein Communicated by M.C. Jordan     

Spatial and temporal patterns of green fluorescent protein (GFP) fluorescence during leaf canopy development in transgenic oilseed rape,<Emphasis Type="Italic"> Brassica napus</Emphasis> L.

The green fluorescent protein (GFP) holds promise as a field-level transgene marker. One obstacle to the use of GFP is fluorescence variability observed within leaf canopies. In growth chamber and field experiments, GFP fluorescence in transgenic oilseed rape (Brassica napus) was shown to be variable at each leaf position over time and among different leaves on the same plant. A leaf had its highest GFP fluorescence after emergence and, subsequently, its fluorescence intensity decreased. GFP fluorescence intensity was directly correlated with the concentration of soluble protein. The concentration of the genetically linked recombinant Bacillus thuringiensis (Bt) cry1Ac endotoxin protein also was examined, and GFP fluorescence was positively correlated with Bt throughout development. The results show that GFP can be used as an accurate transgene marker but that aspects of plant developmental should be taken into account when interpreting fluorescence measurements.Communicated by M.C. Jordan     

Green fluorescent protein is superior to blue fluorescent protein as a quantitative reporter of promoter activity in <Emphasis Type="Italic">E. coli</Emphasis>

Fluorescent proteins related to and derived from green fluorescent protein (GFP) are widely used as tools for investigating a wide range of biological processes. In particular, GFP and its relatives have been used extensively as qualitative reporters of gene expression in many different organisms, but relatively few studies have investigated fluorescent proteins as quantitative reporters of gene expression. GFP has some limitations as a reporter gene, including possible toxicity when expressed at high levels. Therefore, it would be useful if other fluorescent proteins could be identified for use as quantitative reporters. Toward this end, we investigated BFP as a quantitative reporter of promoter activity in E. coli and directly compared it with GFPuv using a set of well-characterized synthetic constitutive promoters. The fluorescence produced in E. coli strains expressing GFPuv or BFP grown on solid medium was quantified using a CCD camera and fluorimetry. GFPuv consistently gave more reliable and statistically significant results than did BFP in all assays. Correspondingly, we found that the signal-to-noise ratio for GFPuv fluorescence is substantially higher than for BFP. We conclude that, under the conditions assessed in this study, GFPuv is superior to BFP as a quantitative reporter of promoter activity in E. coli. J. Bayes, M. Calvey, L. Reineke, A. Colagiavanni, and M. Tscheiner made equivalent contributions to this work.     

Green fluorescent protein as an all-purpose reporter in Petunia

Two critical attributes of a reporter gene are ease of scoring for activity and capacity for expression in all cell types. We have examined a variant of the gene encoding green fluorescent protein,mgfp5, for its ability to meet these criteria in petunia. Under regulation of the Cauliflower Mosaic Virus (CaMV) 35S promoter, GFP was detectable in all vegetative and most floral cell types. Promoters from petuniaadhl andadh2 allowed for production of GFP in those few cell types lacking GFP production from the CaMV 35S promoter, verifying its capacity for expression in all cell types. With the appropriate promoter, GFP fluorescence was thus readily detectable throughout the plant. A potential complication is the green autofluorescence exhibited by some plant tissues. This auto-fluorescence is for the most part distinguishable from that contributed by GFP, but under-scores the need for appropriate controls in GFP-reporter-based experiments. An erratum to this article is available at .     

Protein mislocalization in plant cells using a GFP‐binding chromobody

A key challenge in cell biology is to directly link protein localization to function. The green fluorescent protein (GFP)‐binding protein, GBP, is a 13‐kDa soluble protein derived from a llama heavy chain antibody that binds with high affinity to GFP as well as to some GFP variants such as yellow fluorescent protein (YFP). A GBP fusion to the red fluorescent protein (RFP), a molecule termed a chromobody, was previously used to trace in vivo the localization of various animal antigens. In this study, we extend the use of chromobody technology to plant cells and develop several applications for the in vivo study of GFP‐tagged plant proteins. We took advantage of Agrobacterium tumefaciens‐mediated transient expression assays (agroinfiltration) and virus expression vectors (agroinfection) to express functional GBP:RFP fusion (chromobody) in the model plant Nicotiana benthamiana. We showed that the chromobody is effective in binding GFP‐ and YFP‐tagged proteins in planta. Most interestingly, GBP:RFP can be applied to interfere with the function of GFP fusion protein and to mislocalize (trap) GFP fusions to the plant cytoplasm in order to alter the phenotype mediated by the targeted proteins. Chromobody technology, therefore, represents a new alternative technique for protein interference that can directly link localization of plant proteins to in vivo function.     

Enhancement of organophosphorus hydrolase yield in Escherichia coli using multiple gene fusions

It was previously shown that organophosphorus hydrolase (OPH) expression and purification could be tracked by fluorescence of green fluorescent protein (GFP) when synthesized as an N-terminal fusion with GFP (Cha et al., 2000; Wu et al., 2000). In order to enhance OPH productivity while utilizing the advantage of the reporter protein (GFP), two copies of OPH were cloned in tandem following the gfp(uv) gene (e.g., GFP-OPH(n=2)). Both anti-GFP and anti-OPH Western blots demonstrated that a higher yield was achieved in comparison to the one copy fusion (GFP-OPH). Importantly, the fusion protein was still fluorescent as determined via microscopy. In contrast, a fusion containing two copies of OPH without GFP, and an operon fusion of two OPHs with two independent ribosomal binding sites, did not result in a higher yield than one OPH expressed alone.     

Development of Lactococcus lactis encoding fluorescent proteins,GFP, mCherry and iRFP regulated by the nisin-controlled gene expression system

Fluorescent proteins are useful reporter molecules for a variety of biological systems. We present an alternative strategy for cloning reporter genes that are regulated by the nisin-controlled gene expression (NICE) system. Lactoccocus lactis was genetically engineered to express green fluorescent protein (GFP), mCherry or near-infrared fluorescent protein (iRFP). The reporter gene sequences were optimized to be expressed by L. lactis using inducible promoter pNis within the pNZ8048 vector. Expression of constructions that carry mCherry or GFP was observed by fluorescence microscopy 2 h after induction with nisin. Expression of iRFP was evaluated at 700 nm using an infrared scanner; cultures induced for 6 h showed greater iRFP expression than non-induced cultures or those expressing GFP. We demonstrated that L. lactis can express efficiently GFP, mCherry and iRFP fluorescent proteins using an inducible expression system. These strains will be useful for live cell imaging studies in vitro or for imaging studies in vivo in the case of iRFP.     

Adenovirus-delivered antisense RNA and shRNA exhibit different silencing efficiencies for the endogenous transforming growth factor-beta (TGF-beta) type II receptor

Gene silencing is an essential tool in gene discovery and gene therapy. Traditionally, viral delivery of antisense RNA and, more recently, small interfering RNA (siRNA) molecules in the form of small hairpin RNAs (shRNA) has been used as a strategy to achieve gene silencing. Nevertheless, the enduring challenge is to identify molecules that specifically and optimally silence a given target gene. In this study, we tested a set of adenovirus-delivered antisense RNA fragments and adenovirus-delivered shRNA molecules for their ability to target human transforming growth factor-beta type II receptor (TGFbetaRII). We used a dicistronic reporter, consisting of the coding sequences for TGFbetaRII and green fluorescent protein (GFP) to screen for optimal silencing agents targeting TGFbetaRII. Our results show, for both antisense RNA and shRNA molecules, that their effectiveness in the GFP screen correlated directly with their ability to reduce exogenously expressed TGFbetaRII. Unexpectedly, the antisense RNAs were unable to silence endogenous TGFbetaRII. In contrast, the shRNAs were able to silence endogenous TGFbetaRII. The shRNA that demonstrated the most pronounced effect on the dicistronic TGFbetaRII/GFP reporter reduced endogenous TGFbetaRII protein expression by 70% in A549 cells and reduced TGFbeta signaling by >80% in HeLa cells.     

Green fluorescent protein and factorial approach: an effective partnership for screening the soluble expression of recombinant proteins in Escherichia coli

We report how the combined use of protein expression reporter green fluorescent protein (GFP), and of an incomplete factorial approach (“InFFact”) made of 12 combinations of different states of three expression variables (bacterial strains, culture media and expression temperatures) created a convenient tool for screening the soluble expression of recombinant proteins in Escherichia coli (E. coli).In the first part of this work, we used two recombinant proteins that could be easily detected by Western blotting in the soluble fraction of E. coli lysate in most of the 12 InFFact combinations. When these proteins were fused to GFP and used in the same experiment (“InFFact-GFP”), fluorescence signals proved as sensitive and reliable as those provided by Western blotting. A trend analysis based on Western blot signals or on fluorescence allowed finding expression conditions for successfully scaling up the production of both proteins. Thus, GFP allowed InFFact trend analysis to be performed without gel electrophoresis or Western blotting.In the second part, we compared the results obtained by InFFact and InFFact-GFP when two other recombinant proteins were used which, in contrast with the proteins used in the first part, were barely detectable by Western blotting. Surprisingly, InFFact-GFP but not InFFact was able to find expression conditions for successfully scaling up the production of both proteins, suggesting that GFP could increase the solubility of the fusion partner.In conclusion, GFP allowed InFFact to be performed without gel electrophoresis and with at least the same sensitivity and specificity as that of Western blotting.     

绿色荧光蛋白及其在植物研究中的应用

绿色荧光蛋白(GFP)是海洋生物水母(Aequorea victoria)体内的一种发光蛋白,分子量27kD,由238个氨基酸组成。该蛋白65~67位Ser-Tyr-Gly三种氨基酸环化加氧形成特殊的生色团结构。野生型GFP发光较弱,而且gfp-cDNA含有隐蔽型剪切位点,而加工改造的GFP在植物中能够正常表达并且加强了荧光信号。GFP作为新的报告基因和遗传标记被广泛应用于植物研究之中。     

Use of green fluorescent protein as A non-destructive marker for peanut genetic transformation

Summary The ability to non-destructively visualize transient and stable gene expression has made green fluorescent protein (GFP) a most efficient reporter gene for routine plant transformation studies. We have assessed two fluorescent protein mutants, enhanced GFP (EGFP) and enhanced yellow fluorescent protein (EYFP), under the control of the CaMV35S promoter, for their transient expression efficiencies after particle bombardment of embryogenic cultures of the peanut cultivar, Georgia Green. A third construct (p524EGFP.1) that expressed EGFP from a double 35S promoter with an AMV enhancer sequence also was compared. The brightest and most dense fluorescent signals observed during transient expression were from p524EGFP. 1 and EYFP. Optimized bombardment conditions consisted of 0.6 μm diameter gold particles, 12410 kPa bombardment pressure, 95 kPa vacuum pressure, and pretreatment with 0.4 M mannitol. Bombardments with p524EGFP.1 produced tissue sectors expressing GFP that could be visually selected under the fluorescence microscope over multiple subcultures. Embryogenic lines selected for GFP expression initially may have been chimeric since quantitative analysis of expression sometimes showed an increase when GFP-expressing lines, that also contained a hygromycin-resistance gene, subsequently were cultured on hygromycin. Transformed peanut plants expressing GFP were obtained from lines selected either visually or on hygromycin. Integration of the gfp gene in the genomic DNA of regenerated plants was confirmed by Southern blot hybridization and transmission to progeny.     

Confocal quantification of cis-regulatory reporter gene expression in living sea urchin

Quantification of GFP reporter gene expression at single cell level in living sea urchin embryos can now be accomplished by a new method of confocal laser scanning microscopy (CLSM). Eggs injected with a tissue-specific GFP reporter DNA construct were grown to gastrula stage and their fluorescence recorded as a series of contiguous Z-section slices that spanned the entire embryo. To measure the depth-dependent signal decay seen in the successive slices of an image stack, the eggs were coinjected with a freely diffusible internal fluorescent standard, rhodamine dextran. The measured rhodamine fluorescence was used to generate a computational correction for the depth-dependent loss of GFP fluorescence per slice. The intensity of GFP fluorescence was converted to the number of GFP molecules using a conversion constant derived from CLSM imaging of eggs injected with a measured quantity of GFP protein. The outcome is a validated method for accurately counting GFP molecules in given cells in reporter gene transfer experiments, as we demonstrate by use of an expression construct expressed exclusively in skeletogenic cells.     

Green Fluorescent Protein As a Cell-Labeling Tool and a Reporter of Gene Expression in Transgenic Rainbow Trout

Green fluorescent protein (GFP) has been used as an indicator of transgene expression in living cells and organisms. For testing the utility of GFP in rainbow trout, we microinjected fertilized eggs with four types of supercoiled constructs containing two variants of GFP complementary DNA (S65T and EGFP), driven by two ubiquitous regulatory elements, human cytomegalovirus immediate early enhancer-promoter (CMV) and Xenopus laevis elongation factor 1α enhancer-promoter (EF1). Green fluorescence was first observed at 3 days postfertilization, when the embryo was in the mid-blastula stage. Fluorescence could be detected mosaically in various types of embryonic cells and tissues of swim-up fry. Both the percentage of fluorescent cells and the fluorescence intensity of GFP-expressing cells on blastoderms, measured with a microscopic photometry system, were highest in CMV-EGFP-microinjected embryos. We conclude that GFP is capable of producing detectable fluorescence in rainbow trout, and can be a powerful tool as a cell marker and reporter gene for cold-water fish, and that analysis of GFP expression in living cells is useful for characterizing the activity of cis-elements in vivo. Received December 21, 1998; accepted March 31, 1999.     

Quantitative analysis of gene expression with an improved green fluorescent protein. p6.

The fast and easy in vivo detection predestines the green fluorescent protein (GFP) for its use as a reporter to quantify promoter activities. We have increased the sensitivity of GFP detection 320-fold compared to the wild-type by constructing gfp+, which contains mutations improving the folding efficiency and the fluorescence yield of GFP+. Twelve expression levels were measured using fusions of the gfp+ and lacZ genes with the tetA promoter in Escherichia coli. The agreement of GFP+ fluorescence with beta-galactosidase activities was excellent, demonstrating that the gfp+ gene can be used to accurately quantify gene expression in vivo. However, expression of the gfp+ gene from the stronger hsp60 promoter revealed that high cellular concentrations of GFP+ caused an inner filter effect reducing the fluorescence by 50%, thus underestimating promoter activity. This effect is probably due to the higher absorbance of cells containing GFP+. Thus promoters with activities differing by about two orders of magnitude can be correctly quantified using the gfp+ gene. Possibilities of using GFP variants beyond this range are discussed.     

A Protocol for the Fluorometric Quantification of mGFP5-ER and sGFP(S65T) in Transgenic Plants

The Green Fluorescent Protein (GFP) from Aequorea victoria has begun to be used as a reporter protein in plants. It is particularly useful as GFP fluorescence can be detected in a non-destructive manner, whereas detection of enzyme-based reporters often requires destruction of the plant tissue. The use of GFP as a reporter enables transgenic plant tissues to be screened in vivo at any growth stage. Quantification of GFP in transgenic plant extracts will increase the utility of GFP as a reporter protein. We report herein the quantification of a mGFP5-ER variant in tobacco leaf extracts by UV excitation and a sGFP(S65T) variant in sugarcane leaf and callus extracts by blue light excitation using the BioRad VersaFluor^TM Fluorometer System or the Labsystems Fluoroskan Ascent FL equipped with a narrow band emission filter (510 ± 5 nm). The GFP concentration in transgenic plant extracts was determined from a GFP-standard series prepared in untransformed plant extract with concentrations ranging from 0.1 to 4 g/ml of purified rGFP. Levels of sgfp(S65T) expression, driven by the maize ubiquitin promoter, in sugarcane calli and leaves ranged up to 0.525 g and 2.11 g sGFP(S65T) per mg of extractable protein respectively. In tobacco leaves the expression of mgfp5-ER, driven by the cauliflower mosaic virus (CaMV) 35S promoter, ranged up to 7.05 g mGFP5-ER per mg extractable protein.     

End-joining of reconstituted histone H2AX-containing chromatin in vitro by soluble nuclear proteins from human cells

Green fluorescent protein (GFP) is an attractive reporter for Salmonella gene expression analysis but might interfere with virulence when expressed at high levels. To identify suitable GFP levels, we constructed a series of Salmonella strains expressing different amounts of GFP and measured their fluorescence and colonization levels in infected mice. The results show that GFP concentrations in the range of 7000-200,000 molecules per Salmonella cell are detectable in ex vivo samples using flow cytometry, and cause no major Salmonella virulence defect. Appropriate GFP levels can be obtained with weak promoters and stable GFP, or strong promoters and destabilized GFP.     

Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants

Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the -glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.