Targeting aequorin and green fluorescent protein to intracellular organelles

Two proteins of Aequorea victoria were molecularly engineered and produced in mammalian cells, in order to serve as specific reporters of subcellular microenvironments. Aequorin (AEQ), a Ca²⁺-sensitive photoprotein, was successfully targeted to three intracellular locations: cytosol, nucleus and mitochondria. The recombinant apoprotein, reconstituted into active AEQ by the addition of the prosthetic group to the culture medium, allows the direct measurement of [Ca²⁺] within those compartments, thus directly addressing questions of large biological interest. The same approach was utilized for the green fluorescent protein (GFP) for specific labelling, in vivo, of the various subcellular structures. GFP was targeted to mitochondria: the recombinant protein, strongly fluorescent in a highly reducing environment, provides a powerful tool for visualizing these organelles in living cells, and may represent the prototype of a new family of intracellularly targeted fluorescent probes.     

Display of green fluorescent protein on Escherichia coli cell surface

In this study, expression of green fluorescence protein (GFP) on the external surface of Escherichia coli was achieved by construction of a fusion protein between Lpp-OmpA hybrid and a GFP variant, GFPmut2. The GFP was fused in frame to the carboxyl-terminus of Lpp-OmpA fusion previously shown to direct various other heterologous proteins to E. coli cell surface. Western blot analysis of membrane fractions identified the Lpp-OmpA-GFP fusion protein with the expected size (43 kDa). Immunofluorescence microscopy, immunoelectron microscopy, protease and extracellular pH sensitivity assays further confirmed that GFP is anchored on the outer membrane. The GFP displayed on the E. coli outer surface retained its fluorescence and was not susceptible to the indigenous outer membrane protease OmpT even though there are two putative OmpT proteolytic sites present in GFP. Optimization of the expression conditions was conducted using fluorometry, eliminating cumbersome immuno-labeling procedures. Surface-displayed GFP could be used in a variety of applications including screening of polypeptide libraries, development of live vaccines, construction of whole cell allosteric biosensors, and signal transduction studies.     

Quantitative measurement of green fluorescent protein expression

Summary Data presented here shows a time course analysis of E. coli shake flask cultures expressing the reporter gene green fluorescent protein (GFP) with simultaneous comparison of microbial fluorescence intensity measurements and GFP concentration measured by Western blot. There is an apparent lag between the presence of GFP and its fluorescence due to the time required for formation of the chromophore. We demonstrate that GFP fluorescence can be used as a quantifiable reporter gene, provided the cyclization time for chromophore formation is considered.     

Transient expression of the green fluorescent protein in pollen

 ZM13 is a pollen-specific maize gene which is expressed in the late stages of pollen development. We wished to utilize the ZM13 promoter to examine the expression of a synthetic green fluorescent protein (SGFP) in germinating pollen. The usefulness of the SGFP expression product is that its appearance and distribution can be monitored non-destructively in vivo. A plasmid containing the SGFP coding region under the control of the ZM13 promoter was constructed and then transiently transformed into pollen of Tradescantia paludosa and Nicotiana tabacum by the use of microprojectile bombardment. The expression of the green fluorescent protein was analyzed by fluorescence microscopy using a fluorescein filter. Expression began about 3 h post-bombardment, and all parts of the pollen grain and tube fluoresced. High levels of fluorescence were observed for several days following treatment. Received: 15 February 1998 / Revision accepted: 22 April 1998     

Monitoring the expression of green fluorescent protein in carrot

Green fluorescent protein (GFP) was successfully used as a visual reporter at various stages of carrot (Daucus carota L.) transformation. GFP-fluorescence was non-invasively observed in protoplasts, callus and plants after the delivery of mgfp5-er gene using two transformation methods: direct DNA transfer into polyethylene glycol (PEG) -treated protoplasts and inoculation of root discs with Agrobacterium rhizogenes. Transient GFP-expression was detected in the treated protoplasts and monitored during the first week of the cell culture until the stable level of expression was observed. It was useful for the comparison of protoplast susceptibility to DNA uptake and the transgene expression as the fluorescence declined with various rates depending on the used carrot genotype and PEG-concentration. GFP-monitoring in callus enabled the selection of stably expressing lines. It also allowed verification of the homogeneous tissue composition with regard to the expression of the transgene. In plants, GFP-performance depended on the assayed tissue and organ despite of the constitutive 35S promoter. The expression was visually detected in both vegetative and generative parts, but particularly strong fluorescence was observed in leaf marginal meristems, petioles, stems, and styles. Those tissues can be convenient for examination of the transgenic plants during their growth. The results encourage that GFP is a valuable reporter and can be routinely used for optimization of transformation protocol, selection of transformants and monitoring transgenic carrot.     

GFP在绿色荧光裸鼠不同组织中的表达及分析

目的研究外源绿色荧光蛋白（green fluorescent protein,简称GFP）基因在BALB/c绿色荧光裸鼠主要器官组织中的表达及其差异。方法小动物成像系统和RT-PCR方法检测GFP的组织分布以及荧光表达水平情况。结果经活体荧光影像系统观察及PCR方法检测发现GFP可以在裸鼠多个器官组织中表达,其中在胰腺、心脏、全脑、皮肤、睾丸中表达量较高。结论外源绿色荧光蛋白可以在模型动物体内成功表达且稳定遗传,其中在胰腺组织中高表达。     

High level Purkinje cell specific expression of green fluorescent protein in transgenic mice

The green fluorescent protein (GFP) has become a powerful tool in molecular and cell biology. It is a commonly used marker for cloning and transfection experiments as well as a useful label of living cells allowing continuous observation of developing structures. In order to unravel mechanisms of neuronal differentiation, we generated a transgenic mouse model which expresses GFPS65T,hu under the control of the Purkinje cell-specific promoter L7/pcp-2. Here, we show that GFPS65T,hu is highly expressed specifically in the cerebellum in whole mount preparations after the 2nd postnatal week. GFPS65T,hu can be detected exclusively in Purkinje cells of cerebellar slices. The fluorescence intensity of GFPS65T,hu should enable the characterization and recording of axons, dendrites, and spines protruding from these neuronal processes. The level of GFP expression can be quantified by western blotting which allows to analyze protein expression and L7/pcp-2 promoter regulation in vivo. The application of cellular and physiological techniques on L7GFP mice will provide a remarkable opportunity to investigate various aspects of neuronal development at the cellular and subcellular levels.     

Targeting gene-modified hematopoietic cells to the central nervous system: use of green fluorescent protein uncovers microglial engraftment.

Gene therapy in the central nervous system (CNS) is hindered by the presence of the blood-brain barrier, which restricts access of serum constituents and peripheral cells to the brain parenchyma. Expression of exogenously administered genes in the CNS has been achieved in vivo using highly invasive routes, or ex vivo relying on the direct implantation of genetically modified cells into the brain. Here we provide evidence for a novel, noninvasive approach for targeting potential therapeutic factors to the CNS. Genetically-modified hematopoietic cells enter the CNS and differentiate into microglia after bone-marrow transplantation. Up to a quarter of the regional microglial population is donor-derived by four months after transplantation. Microglial engraftment is enhanced by neuropathology, and gene-modified myeloid cells are specifically attracted to the sites of neuronal damage. Thus, microglia may serve as vehicles for gene delivery to the nervous system.     

High expression of green fluorescent protein in Pichia pastoris leads to formation of fluorescent particles

Wild type gene for green fluorescent protein (GFP) was stably integrated into the Pichia pastoris genome and yielded an expression level of over 40% of total cellular protein. The high cytoplasmic concentration of fluorescent (properly folded and processed) GFP caused the formation of fluorescent spherical structures, which could be observed by fluorescence or confocal microscopy after controlled permeabilization of the yeast cells with 0.2% N-lauroyl sarcosine (NLS). Fluorescent GFP particles were also isolated after removal of the cell wall and found to be quite resistant to 0.2% N-lauroyl sarcosine. SDS-PAGE analysis of the isolated fluorescent particles revealed the presence of an 80 kDa protein (alcohol oxidase) and GFP (30%). We conclude that GFP is able to enter spontaneously into the peroxisomes and is inserted into densely packed layers of alcohol oxidase. Consequently, the formation of similar fluorescent particles can also be expected in other organisms when using high-level expression systems. As GFP is widely used in fusion with other proteins as a reporter for protein localization and for many other applications in biotechnology, care must be taken to avoid false interpretations of targeting or trafficking mechanisms inside the cells. In addition, when whole cells or cytoplasmic fractions are used for the quantitative determination of GFP levels, incorrect and misleading values of GFP could be obtained due to the formation of fluorescent particles containing material inside which is not available for fluorescence measurements.     

Patterns of green fluorescent protein expression in transgenic plants

Modified forms of genes encoding green fluorescent protein (GFP) can be macroscopically detected when expressed in whole plants. This technology has opened up new uses for GFP such as monitoring transgene presence and expression in the environment once it is linked or fused to a gene of interest. When whole-plant or whole-organ GFP visualization is required, GFP should be predictably expressed and reliably fluorescent. In this study the whole plant expression and fluorescence patterns of a mGFP5er gene driven by the cauliflower mosaic virus 35S promoter was studied in intact GFP-expressing transgenic tobacco (Nicotiana tabacum cv. Xanthi). It was shown that GFP synthesis levels in single plant organs were similar to GUS activity levels from published data when driven by the same promoter. Under the control of the 35S promoter, high expression of GFP can be used to visualize stems, young leaves, flowers, and organs where the 35S promoter is most active. Modified forms of GFP could replace GUS as the visual marker gene of choice.     

Influence of cell surface structures on crenarchaeal biofilm formation using a thermostable green fluorescent protein

The thermoacidophilic crenarchaeote Sulfolobus acidocaldarius displays three distinct type IV pili-like structures on its surface: (i) the flagellum, (ii) the UV-induced pili and (iii) the adhesive pili. In bacteria, surface appendages play an important role in the spatial organization of cells from initial surface attachment to the development of mature community structures. To investigate the influence of the diverse set of type IV pili-like structures in S. acidocaldarius, single, double and triple mutants lacking the cell surface appendages were constructed and analysed for their behaviour in attachment assays and during biofilm formation. A heat stable green fluorescent protein was employed the first time in a hyperthermophilic archaeon. A codon adjusted eCGP123 was expressed to study mixed biofilms of different deletion mutants to understand the interplay of the surface structures during biofilm formation. During this process the deletion of the adhesive pili and UV-induced pili led to the most pronounced effects, either an increase in cell density or increased cluster formation respectively. However, all three cell surface appendages played a role in the colonization of surfaces and only the interplay of all three appendages leads to the observed wild-type biofilm phenotype.     

Reliable use of green fluorescent protein in fluorescent pseudomonads.

When fluorescent pseudomonads are cultured on standard solid media under iron limiting conditions, they produce fluorescent, pigmented iron collating agents (siderophores). Siderophores can be readily identified by strong fluorescence seen under UV/blue light. The application of the eukaryotic green fluorescent protein (GFP) as a bacterial marker in microbial ecology is increasingly being used, particularly as it is a powerful method for non-destructive monitoring in situ. As gfp expressing bacteria have to be detected under UV/blue light, the fluorescence of siderophore-producing Pseudomonas spp. masks normal levels of GFP fluorescence when colonies are viewed on standard bacterial agar. Here, we describe a simple but effective way of identifying gfp-expressing Pseudomonas fluorescens using media supplemented with 0.45 mM FeSO(4).7H(2)O. This is of relevance for the screening of insertion libraries and in the application of GFP transposons as promoter probes.     

Differentiation of embryonic stem cell to astrocytes visualized by green fluorescent protein

Green fluorescent protein (GFP) gene was transfected and expressed in murine embryonic stem (ES) cells under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. Stably transfected cells were characterized by immunohistochemistry and by fluorescence microscopy. Cells containing GFP were differentiated to Type I and Type II astrocytes after induction by all-trans retinoic acid. Differentiated cells were expressed GFP and visualized by fluorescence microscopy. Differentiated cells expressed GFP were correlated with the expression of GFAP and morphological change. It demonstrates that the cell line expressed GFP can be used to trace the morphological changes of astrocytes during differentiation, and further for the isolation of astrocytes from the mixed cells differentiated from ES cell.     

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.     

Circularly permuted variants of the green fluorescent protein.

Folding of the green fluorescent protein (GFP) from Aequorea victoria is characterized by autocatalytic formation of its p-hydroxybenzylideneimidazolidone chromophore, which is located in the center of an 11-stranded beta-barrel. We have analyzed the in vivo folding of 20 circularly permuted variants of GFP and find a relatively low tolerance towards disruption of the polypeptide chain by introduction of new termini. All permuted variants with termini in strands of the beta-barrel and about half of the variants with termini in loops lost the ability to form the chromophore. The thermal stability of the permuted GFPs with intact chromophore is very similar to that of the wild-type, indicating that chromophore-side chain interactions strongly contribute to the extraordinary stability of GFP.     

Charge transfer in green fluorescent protein.

Charge transfer reactions that contribute to the photoreactions of the wild type green fluorescent protein (GFP) do not occur in the isolated p-hydroxybenzylidene-imidazolidinone chromophore, demonstrating the role of the protein environment. The high quantum efficiency of the fluorescence photocycle that includes excited state proton transfer and the suppression of non-radiative pathways by the protein environment have been correlated with structural dynamics in the chromophore environment. A low quantum efficiency competing phototransformation reaction of GFP is accompanied by both proton and electron transfer, and closely mimics the charge redistribution that is occurring in the fluorescence photocycle. The protein response to this destabilising event has been demonstrated by cryo-trapping of early products in the reaction pathway and is found to be strong even at 100 K, including displacements of chromophore, protein, solvent and a photogenerated CO2 molecule derived from the decarboxylated Glu 222 side chain. We discuss the ramifications of the observation of strong conformational perturbations below the protein dynamical transition at approximately 200 K, in view of low temperature work on other light sensitive proteins such as myoglobin and bacteriorhodopsin. The proton and electron transfer in the phototransformation pathway mimics the proton and charge transfer which occurs during the fluorescence cycle, which leads to common structural responses in both photoreactions as shown by ultrafast spectroscopy. We review and discuss literature on light-induced and thermal charge transfer events, focusing on recent findings addressing conformational dynamics and implications for thermodynamic properties.     

Functional expression of green fluorescent protein derivatives in Halobacterium salinarum

We investigated the applicability of the green fluorescent protein (GFP) of Aequorea victoria as a reporter for gene expression in an extremely halophilic organism: Halobacterium salinarum. Two recombinant GFPs were fused with bacteriorhodopsin, a typical membrane protein of H. salinarum. These fusion proteins preserved the intrinsic functions of each component, bacteriorhodopsin and GFP, were expressed in H. salinarum under conditions with an extremely high salt concentration, and were proved to be properly localized in its plasma membrane. These results suggest that GFP could be used as a versatile reporter of gene expression in H. salinarum for investigations of various halophilic membrane proteins, such as sensory rhodopsin or phoborhodopsin.     

Analysis with flow cytometry of green fluorescent protein expression in leukemic cells.

BACKGROUND: The measurement of DNA content with propidium iodide (PI) in cells transfected with expression vectors encoding the green fluorescent protein (GFP) is a useful tool in studying a variety of biological functions of proteins within cells. The purpose of this study was to determine conditions of formaldehyde fixation that permit intracellular GFP fluorescence and adequate DNA histograms to be generated following transient transfection of cells with a GFP-encoding plasmid. Cell cycle analysis was also performed in GFP-positive cells. METHODS: The murine myeloid leukemic cell line, 32Dcl3, was used as the model system. Cells were transfected with a GFP-encoding plasmid (pEGFPC1). Following fixation in different formaldehyde concentrations and permeabilization with 70% ethanol, cells were stained with PI and analyzed by flow cytometry for GFP fluorescence and DNA content. Transfected cells were also analyzed for GFP fluorescence and DNA content following release from nocodazole block. RESULTS: Fixing cells in 0.51-1.75% formaldehyde concentrations prior to ethanol permeabilization resulted in 14-19% of transfected cells being GFP-positive, with acceptable coefficients of variation on the G(1) peak of DNA histograms. Analysis of cells synchronized to and released from the G(2)-M phase by nocodazole suggested that GFP-positive cells, when compared to GFP-negative cells, did not appear to progress out of G(2)-M following release from nocodazole block. Simultaneous detection of GFP fluorescence and DNA content by PI staining is possible following transient transfection of cells with a single expression vector encoding GFP. Our results demonstrate that GFP expression can be detected, using flow cytometry to perform cell cycle analysis in murine leukemic cells.     

Differential rates of gene expression monitored by green fluorescent protein

The use of green fluorescent protein (GFP) as a reporter gene has made a broad impact in several areas, especially in studies of protein trafficking, localization, and expression analysis. GFP's many advantages are that it is small, autocatalytic, and does not require fixation, cell disruption, or the addition of cofactors or substrates. Two characteristics of GFP, extreme stability and chromophore cyclization lag time, pose a hindrance to the application of GFP as a real-time gene expression reporter in bioprocess applications. In this report, we present analytical methods that overcome these problems and enable the temporal visualization of discrete gene regulatory events. The approach we present measures the rate of change in GFP fluorescence, which in turn reflects the rate of gene expression. We conducted fermentation and microplate experiments using a protein synthesis inhibitor to illustrate the feasibility of this system. Additional experiments using the classic gene regulation of the araBAD operon show the utility of GFP as a near real-time indicator of gene regulation. With repetitive induction and repression of the arabinose promoter, the differential rate of GFP fluorescence emission shows corresponding cyclical changes during the culture.