Recent Advances in GFP Folding Reporter and Split-GFP Solubility Reporter Technologies. Application to Improving the Folding and Solubility of Recalcitrant Proteins from Mycobacterium tuberculosis

We have improved our green fluorescent protein (GFP) folding reporter technology [Waldo et al., (1999) Nat. Biotechnol. 17, 691–695] to evolve recalcitrant proteins from Mycobacterium tuberculosis. The target protein is inserted into the scaffolding of the GFP, eliminating false-positive artifacts caused by expression of truncated protein variants from internal cryptic ribosome binding sites in the target RNA. In parallel, we have developed a new quantitative fluorescent protein tagging and detection system based on micro-domains of GFP. This split-GFP system, which works both in vivo and in vitro, is amenable to high-throughput assays of protein expression and solubility [Cabantous et al., (2005) Nat. Biotechnol. 23, 102–107]. Together, the GFP folding reporter and split-GFP technologies offer a comprehensive system for manipulating and improving protein folding and solubility.     

A green fluorescent protein fusion strategy for monitoring the expression, cellular location, and separation of biologically active organophosphorus hydrolase

 Organophosphorus hydrolase (OPH) is capable of degrading a variety of pesticides and nerve agents. We have developed a versatile monitoring technique for detecting the amount of OPH during the expression and purification steps. This involves fusion of the gene for green fluorescent protein (GFP) to the 5′ end of the OPH gene and subsequent expression in Escherichia coli. The synthesized fusion protein was directly visualized due to the optical properties of GFP. Western blot analyses showed that the correct fusion protein was expressed after IPTG-induction. Also, the in vivo GFP fluorescence intensity was proportional to the OPH enzyme activity. Moreover, the OPH, which forms a dimer in its active state, retained activity while fused to GFP. Enterokinase digestion experiments showed that OPH was separated from the GFP reporter after purification via immobilized metal affinity chromatography, which in turn was monitored by fluorescence. The strategy of linking GFP to OPH has enormous potential for improving enzyme production efficiency, as well as enhancing field use, as it can be monitored at low concentrations with inexpensive instrumentation based on detecting green fluorescence. Received: 27 April 1999 / Received last revision: 18 October 1999 / Accepted: 1 November 1999     

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.     

Construction of an engineered yeast with glucose-inducible emission of green fluorescence from the cell surface

An engineered yeast with emission of fluorescence from the cell surface was constructed. Cell surface engineering was applied to display a visible reporter molecule, green fluorescent protein (GFP). A glucose-inducible promoter GAPDH as a model promoter was selected to control the expression of the reporter gene in response to environmental changes. The GFP gene was fused with the gene encoding the C-terminal half of α-agglutinin of Saccharomyces cerevisiae having a glycosylphosphatidylinositol anchor attachment signal sequence. A secretion signal sequence of the fungal glucoamylase precursor protein was connected to the N-terminal of GFP. This designed gene was integrated into the TRP1 locus of the chromosome of S. cerevisiae with homologous recombination. Fluorescence microscopy demonstrated that the transformant cells emitted green fluorescence derived from functionally expressed GFP involved in the fusion molecule. The surface display of GFP was further verified by immunofluorescence labeling with a polyclonal antibody (raised in rabbits) against GFP as the first antibody and Rhodamine Red-X-conjugated goat anti-rabbit IgG as the second antibody which cannot penetrate into the cell membrane. The display of GFP on the cell surface was confirmed using a confocal laser scanning microscope and by measuring fluorescence in each cell fraction obtained after the subcellular fractionation. As GFP was proved to be displayed as an active form on the cell surface, selection of promoters will endow yeast cells with abilities to respond to changes in environmental conditions, including nutrient concentrations in the media, through the emission of fluorescence. Received: 23 August 1999 / Received revision: 16 November 1999 / Accepted: 29 November 1999     

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.     

Mutations that suppress the thermosensitivity of green fluorescent protein

Background The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has recently attracted great interest as the first example of a cloned reporter protein that is intrinsically fluorescent. Although successful in some organisms, heterologous expression of GFP has not always been straight forward. In particular, expression of GFP in cells that require incubation temperatures around 37°C has been problematic.Results We have carried out a screen for mutant forms of GFP that fluoresce more intensely than the wild-type protein when expressed in E. coli at 37°C. We have characterized a bright mutant (GFPA) with reduced sensitivity to temperature in both bacteria and yeast, and have shown that the amino acids substituted in GFPA act by preventing temperature-dependent misfolding of the GFP apoprotein. We have shown that the excitation and emission spectra of GFPA can be manipulated by site-directed mutagenesis without disturbing its improved folding characteristics, and have produced a thermostable folding mutant (GFP5) that can be efficiently excited using either long-wavelength ultraviolet or blue light. Expression of GFP5 results in greatly improved levels of fluorescence in both microbial and mammalian cells cultured at 37°C.Conclusions The thermotolerant mutants of GFP greatly improve the sensitivity of the protein as a visible reporter molecule in bacterial, yeast and mammalian cells. The fluorescence spectra of these mutants can be manipulated by further mutagenesis without deleteriously affecting their improved folding characteristics, so it may be possible to engineer a range of spectral variants with improved tolerance to temperature. Such a range of sensitive reporter proteins will greatly improve the prospects for GFP-based applications in cells that require relatively high incubation temperatures.     

Transient expression and stable transformation of soybean using the jellyfish green fluorescent protein

 Embryogenic soybean [Glycine max (L.) Merrill.] suspension cultures were bombarded with five different gene constructions encoding the jellyfish (Aequorea victoria) green fluorescent protein (GFP). These constructions had altered codon usage compared to the native GFP gene and mutations that increased the solubility of the protein and/or altered the native chromophore. All of the constructions produced green fluorescence in soybean cultures upon blue light excitation, although a soluble modified red-shifted GFP (smRS-GFP) was the easiest to detect based on the brightness and number of foci produced. Expression of smRS-GFP was visible as early as 1.5 h after bombardment, with peak expression at approximately 6.5 h. Large numbers of smRS-GFP-expressing areas were visible for 48 h postbombardment and declined rapidly thereafter. Stably transformed cultures and plants exhibited variation in the intensity and location of GFP expression. PCR and Southern hybridization analyses confirmed the presence of introduced GFP genes in stably transformed cultures. Received: 23 September 1998 / Revision received: 4 January 1999 / Accepted: 15 January 1999     

Depolarization after resonance energy transfer (DARET): a sensitive fluorescence-based assay for botulinum neurotoxin protease activity

The DARET (depolarization after resonance energy transfer) assay is a coupled Förster resonance energy transfer (FRET)–fluorescence polarization assay for botulinum neurotoxin type A or E (BoNT/A or BoNT/E) proteolytic activity that relies on a fully recombinant substrate. The substrate consists of blue fluorescent protein (BFP) and green fluorescent protein (GFP) flanking SNAP-25 (synaptosome-associated protein of 25 kDa) residues 134–206. In this assay, the substrate is excited with polarized light at 387 nm, which primarily excites the BFP, whereas emission from the GFP is monitored at 509 nm. Energy transfer from the BFP to the GFP in the intact substrate results in a substantial depolarization of the GFP emission. The energy transfer is eliminated when the fluorescent domains separate on cleavage by the endopeptidase, and emission from the directly excited GFP product fragment is then highly polarized, resulting in an overall increase in polarization. This increase in polarization can be monitored to assay the proteolytic activity of BoNT/A and BoNT/E in real time. It allows determination of the turnover rate of the substrate and the kinetic constants (V_max and k_cat) based on the concentration of cleaved substrate determined directly from the measurements using the additivity properties of polarization. The assay is amenable to high-throughput applications.     

表达GFP/mCherry白色念珠菌的构建及其在与巨噬细胞互作中的应用

白色念珠菌(Candida albicans)被巨噬细胞吞噬的效率与被吞噬后的形态观察是研究白色念珠菌与巨噬细胞互作的重要内容。【目的】以野生型菌株SC5314为母本,构建能够表达绿色荧光蛋白(green fluorescent protein, GFP)/mCherry的白色念珠菌,应用于巨噬细胞与白色念珠菌互作的研究。【方法】通过生长与形态观察、细胞活性检测及小鼠系统性感染模型确定荧光蛋白的表达对菌株生长、形态与毒力的影响;在共培养条件下,通过流式细胞术及荧光显微镜检测巨噬细胞的吞噬率及白色念珠菌的形态变化。【结果】构建的菌株在表型上与野生型菌株一致,并可用于在共培养下测定巨噬细胞吞噬率的流式细胞术以及观察白色念珠菌的形态变化。【结论】表达荧光蛋白的菌株为研究巨噬细胞与白色念珠菌的互作提供了新方法。     

Nanoscopy in a living multicellular organism expressing GFP

We report superresolution fluorescence microscopy in an intact living organism, namely Caenorhabditis elegans nematodes expressing green fluorescent protein (GFP)-fusion proteins. We also superresolve, by stimulated emission depletion (STED) microscopy, living cultured cells, demonstrating that STED microscopy with GFP can be widely applied. STED with GFP can be performed with both pulsed and continuous-wave lasers spanning a wide wavelength range from at least 556–592 nm. Acquiring subdiffraction resolution images within seconds enables the recording of movies revealing structural dynamics. These results demonstrate that numerous microscopy studies of live samples employing GFP as the marker can be performed at subdiffraction resolution.     

A family of GFP-like proteins with different spectral properties in lancelet <Emphasis Type="Italic">Branchiostoma floridae</Emphasis>

Background  

Members of the green fluorescent protein (GFP) family share sequence similarity and the 11-stranded β-barrel fold. Fluorescence or bright coloration, observed in many members of this family, is enabled by the intrinsic properties of the polypeptide chain itself, without the requirement for cofactors. Amino acid sequence of fluorescent proteins can be altered by genetic engineering to produce variants with different spectral properties, suitable for direct visualization of molecular and cellular processes. Naturally occurring GFP-like proteins include fluorescent proteins from cnidarians of the Hydrozoa and Anthozoa classes, and from copepods of the Pontellidae family, as well as non-fluorescent proteins from Anthozoa. Recently, an mRNA encoding a fluorescent GFP-like protein AmphiGFP, related to GFP from Pontellidae, has been isolated from the lancelet Branchiostoma floridae, a cephalochordate (Deheyn et al., Biol Bull, 2007 213:95).     

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.     

Use of green fluorescent protein for detection of transformed shoots and homozygous offspring

 Using tobacco as a model system, the data obtained demonstrated that the green fluorescent protein (GFP) can be used as a visual selection marker for transformed tissues. Based on differences in the intensity of GFP fluorescence, homozygous and hemizygous states could be easily visualized in seeds and seedlings of the T1 generation. These results were confirmed by genetic analysis. Optimized conditions for GFP analysis of stable transformants are presented. Received: 9 February 1999 / Revision received: 26 April 1999 / Accepted: 19 May 1999     

Promoters derived from banana bunchy top virus DNA-1 to −5 direct vascular-associated expression in transgenic banana (Musa spp.)

The intergenic regions of banana bunchy top virus (BBTV) DNA-1 to -5 were fused to the green fluorescent protein (GFP) and uidA reporter genes and assessed for promoter activity in transgenic banana (Musa spp. cv. Bluggoe). Promoter activity associated with the BBTV-derived promoters was transgene dependent with greatest activity observed using the GFP reporter. The BBTV promoters (BT1 to BT5) directed expression primarily in vascular-associated cells, although levels of activity varied between individual promoters. Promoters BT4 and BT5 directed the highest levels of GFP expression, while activity from BT1, BT2 and BT3 promoters was considerably weaker. Intron-mediated enhancement, using the maize polyubiquitin 1 (ubi1) intron, generated a significant increase in GUS expression directed by the BBTV promoters in transgenic plants. Received: 17 May 1999 / Revision received: 3 November 1999 / Accepted: 4 November 1999     

Evaluation of GFP Tag as a Screening Reporter in Directed Evolution of a Hyperthermophilic β-Glucosidase

By applying a directed evolution methodology specific enzymatic characteristics can be enhanced, but to select mutants of interest from a large mutant bank, this approach requires high throughput screening and facile selection. To facilitate such primary screening of enhanced clones, an expression system was tested that uses a green fluorescent protein (GFP) tag from Aequorea victoria linked to the enzyme of interest. As GFP’s fluorescence is readily measured, and as there is a 1:1 molar correlation between the target protein and GFP, the concept proposed was to determine whether GFP could facilitate primary screening of error-prone PCR (EPP) clones. For this purpose a thermostable β-glucosidase (BglA) from Fervidobacterium sp. was used as a model enzyme. A vector expressing the chimeric protein BglA-GFP-6XHis was constructed and the fusion protein purified and characterized. When compared to the native proteins, the components of the fusion displayed modified characteristics, such as enhanced GFP thermostability and a higher BglA optimum temperature. Clones carrying mutant BglA proteins obtained by EPP, were screened based on the BglA/GFP activity ratio. Purified tagged enzymes from selected clones resulted in modified substrate specificity.     

Identification of GFP-like Proteins in Nonbioluminescent,Azooxanthellate Anthozoa Opens New Perspectives for Bioprospecting

We screened nonbioluminescent, azooxanthellate cnidaria as potential sources for advanced marker proteins and succeeded in cloning a tetrameric green fluorescent protein (GFP) from the tentacles of Cerianthus membranaceus. The fluorescence of this protein (cmFP512) is characterized by excitation maximum at 503 nm, emission maximum at 512 nm, extinction coefficient of 58,800 M^–1 cm^–1, quantum yield of 0.66, and fluorescence lifetime of 2.4 ns. The chromophore is formed from the tripeptide Gln-Tyr-Gly. The amino acid sequence of this protein shares 17.8% identical residues with GFP from Aequorea victoria. Weak interactions between the subunits of the tetramer make cmFP512 a promising lead structure for the generation of monomeric variants of fluorescent proteins. Both red fluorescent proteins and nonfluorescent proteins of the GFP family were also purified from tissue homogenates of Adamsia palliata and Calliactis parasitica. The results presented here indicate that a photoprotective function of GFP-like proteins is unlikely in the examined anthozoa species.     

Movement of protein and macromolecules between host plants and the parasitic weed Phelipanche aegyptiaca Pers.

Little is known about the translocation of proteins and other macromolecules from a host plant to the parasitic weed Phelipanche spp. Long-distance movement of proteins between host and parasite was explored using transgenic tomato plants expressing green fluorescent protein (GFP) in their companion cells. We further used fluorescent probes of differing molecular weights to trace vascular continuity between the host plant and the parasite. Accumulation of GFP was observed in the central vascular bundle of leaves and in the root phloem of transgenic tomato plants expressing GFP under the regulation of AtSUC2 promoter. When transgenic tomato plants expressing GFP were parasitized with P. aegyptiaca, extensive GFP was translocated from the host phloem to the parasite phloem and accumulated in both Phelipanche tubercles and shoots. No movement of GFP to the parasite was observed when tobacco plants expressing GFP targeted to the ER were parasitized with P. aegyptiaca. Experiments using fluorescent probes of differing molecular weights to trace vascular continuity between the host plant and the parasite demonstrated that Phelipanche absorbs dextrans up to 70 kDa in size from the host and that this movement can be bi-directional. In the present study, we prove for the first time delivery of proteins from host to the parasitic weed P. aegyptiaca via phloem connections, providing information for developing parasite resistance strategies.     

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     

Red fluorescent Xenopus laevis: a new tool for grafting analysis

Background  

Fluorescent proteins such as the green fluorescent protein (GFP) have widely been used in transgenic animals as reporter genes. Their use in transgenic Xenopus tadpoles is especially of interest, because large numbers of living animals can easily be screened. To track more than one event in the same animal, fluorescent markers that clearly differ in their emission spectrum are needed.     

Microinjection of heme oxygenase genes rescues phytochrome-chromophore-deficient mutants of the moss Ceratodon purpureus

 In protonemal tip cells of the moss Ceratodon purpureus (Hedw.) Brid., phototropism and chlorophyll accumulation are regulated by the photoreceptor phytochrome. The mutant ptr116 lacks both responses as a result of a defect in the biosynthesis of phytochromobilin, the chromophore of phytochrome, at the point of biliverdin formation. The rescue of the phototropic response and of chlorophyll synthesis were tested by injecting different substances into tip cells of ptr116. Microinjection was first optimised with the use of fluorescent dyes and an expression plasmid containing a green fluorescent protein (GFP) gene. Injected phycocyanobilin, which substitutes for phytochromobilin, rescued both the phototropic response and light-induced chlorophyll accumulation in ptr116. The same results were obtained when expression plasmids with heme oxygenase genes of rat (HO-1) and Arabidopsis thaliana (L.) Heynh. (HY1) were injected. Heme oxygenase catalyses the conversion of heme into biliverdin. Whereas HY1 has a plastid target sequence and is presumably transferred to plastids, HO-1 is proposed to be cytosolic. The data show that ptr116 lacks heme oxygenase enzyme activity and indicate that heme oxygenases of various origin are active in Ceratodon bilin synthesis. In addition, it can be inferred from the data that the intracellular localisation of the expressed heme oxygenase is not important since the plastid enzyme can be replaced by a cytosolic one. Received: 8 March 1999 / Accepted: 30 July 1999