Green fluorescent protein – a bright idea for the study of bacterial protein localization

Use of the green fluorescent protein (GFP) of Aequorea victoria as a reporter for protein and DNA localization has provided sensitive, new approaches for studying the organization of the bacterial cell, leading to new insights into diverse cellular processes. GFP has many characteristics that make it useful for localization studies in bacteria, primarily its ability to fluoresce when fused to target polypeptides without the addition of exogenously added substrates. As an alternative to immunofluorescence microscopy, the expression of gfp gene fusions has been used to probe the function of cellular components fundamental for DNA replication, translation, protein export, and signal transduction, that heretofore have been difficult to study in living cells. Moreover, protein and DNA localization can now be monitored in real time, revealing that several proteins important for cell division, development and sporulation are dynamically localized throughout the cell cycle. The use of additional GFP variants that permit the labeling of multiple components within the same cell, and the use of GFP for genetic screens, should continue to make this a valuable tool for addressing complex questions about the bacterial cell.     

Rivoflavin may interfere with on-line monitoring of secreted green fluorescence protein fusion proteins in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Background  

Together with the development of optical sensors, fluorometry is becoming an increasingly attractive tool for the monitoring of cultivation processes. In this context, the green fluorescence protein (GFP) has been proposed as a molecular reporter when fused to target proteins to study their subcellular localization or secretion behaviour. The present work evaluates the use of the GFP fusion partner for monitoring extracellular production of a Rhizopus oryzae lipase (ROL) in Pichia pastoris by means of 2D-fluorimetric techniques     

Searching for new cell wall protein genes in Arabidopsis

The objective of this study was to test an approach that combines bioinformatic and subcellular localization analysis to identify novel cell wall protein genes in Arabidopsis. Proteins with unknown function in the Arabidopsis genome were first identified and scanned for the presence of N-terminal signal peptides. The signal peptide-containing function-unknown proteins were further analyzed to eliminate the ones containing other sequences, such as endoplasmic reticulum and vacuole retention signals, that may prevent a protein from secretion into cell walls. The top ten genes passing the bioinformatic analysis were selected for protein subcellular localization using green fluorescence protein (GFP) as a reporter. A vector was constructed for high throughput gene-GFP fusion protein generation and overexpression in Arabidopsis for gene function analysis. Transformants of six genes showed reasonable expression of GFP fusion protein. However, none of the transformants showed GFP localization in cell walls. The low rate of new cell wall protein discovery suggests that the number of unidentified cell wall proteins in the Arabidopsis genome may be small.     

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.     

Purification of GFP fusion proteins with high purity and yield by monoclonal antibody-coupled affinity column chromatography

GFP has often been used as a marker of gene expression, protein localization in living and fixed tissues as well as for protein targeting in intact cells and organisms. Monitoring foreign protein expression via GFP fusion is also very appealing for bioprocess applications. Many cells, including bacterial, fungal, plant, insect and mammalian cells, can express recombinant GFP (rGFP) efficiently. Several methods and procedures have been developed to purify the rGFP or recombinant proteins fused with GFP tag. However, most current GFP purification methods are limited by poor yields and low purity. In the current study, we developed an improved purification method, utilizing a FMU-GFP.5 monoclonal antibody (mAb) to GFP together with a mAb-coupled affinity chromatography column. The method resulted in a sample that was highly pure (more than 97% homogeneity) and had a sample yield of about 90%. Moreover, the GFP epitope permitted the isolation of almost all the active recombinant target proteins fused with GFP, directly and easily, from the crude cellular sources. Our data suggests this method is more efficient than any currently available method for purification of GFP protein.     

The role of quarternary structure in fluorescent protein stability

The stability of fluorescent proteins (FPs) is of great importance for their use as reporters in studies of gene expression, protein dynamics and localization in cell. A comparative analysis of conformational stability of fluorescent proteins, having different association state was done. The list of studied proteins includes EGFP (monomer of green fluorescent protein, GFP), zFP506 (tetramer GFP), mRFP1 and "dimer2" (monomer and dimmer of red fluorescent protein), DsRed1 (red tetramer). The character of fluorescence intensity changes induced by guanidine hydrochloride (GdnHCl) of these proteins differs significantly. Green tetramer zFP506 has been shown to be more stable than green monomer EGFP, red dimmer "dimer2" has been shown to be less stable than red tetramer DsRed1, while red monomer mRFP1 has been shown to be practically as stable as tetramer DsRedl. It is concluded that the quaternary structure, being an important stabilizing factor, does not represent the only circumstance dictating the dramatic variations between fluorescent proteins in their conformational stability.     

Understanding the folding of GFP using biophysical techniques

Green fluorescent protein (GFP) and its many variants are probably the most widely used proteins in medical and biological research, having been extensively engineered to act as markers of gene expression and protein localization, indicators of protein–protein interactions and biosensors. GFP first folds, before it can undergo an autocatalytic cyclization and oxidation reaction to form the chromophore, and in many applications the folding efficiency of GFP is known to limit its use. Here, we review the recent literature on protein engineering studies that have improved the folding properties of GFP. In addition, we discuss in detail the biophysical work on the folding of GFP that is beginning to reveal how this large and complex structure forms.     

Protein networks markedly improve prediction of subcellular localization in multiple eukaryotic species

The function of a protein is intimately tied to its subcellular localization. Although localizations have been measured for many yeast proteins through systematic GFP fusions, similar studies in other branches of life are still forthcoming. In the interim, various machine-learning methods have been proposed to predict localization using physical characteristics of a protein, such as amino acid content, hydrophobicity, side-chain mass and domain composition. However, there has been comparatively little work on predicting localization using protein networks. Here, we predict protein localizations by integrating an extensive set of protein physical characteristics over a protein's extended protein-protein interaction neighborhood, using a classification framework called 'Divide and Conquer k-Nearest Neighbors' (DC-kNN). These predictions achieve significantly higher accuracy than two well-known methods for predicting protein localization in yeast. Using new GFP imaging experiments, we show that the network-based approach can extend and revise previous annotations made from high-throughput studies. Finally, we show that our approach remains highly predictive in higher eukaryotes such as fly and human, in which most localizations are unknown and the protein network coverage is less substantial.     

A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells

Green fluorescent protein (GFP) and GFP-like proteins of different colors are important tools in cell biology. In many studies, the intracellular targeting of proteins has been determined by transiently expressing GFP fusion proteins and analyzing their intracellular localization by fluorescence microscopy. In most vectors, expression of GFP is driven by the enhancer/promoter cassette of the immediate early gene of human cytomegalovirus (hCMV). This cassette generates high levels of protein expression in most mammalian cell lines. Unfortunately, these nonphysiologically high protein levels have been repeatedly reported to artificially alter the intracellular targeting of proteins fused to GFP. To cope with this problem, we generated a multitude of attenuated GFP expression vectors by modifying the hCMV enhancer/promoter cassette. These modified vectors were transiently expressed, and the expression levels of enhanced green fluorescent protein (EGFP) alone and enhanced yellow fluorescent protein (EYFP) fused to another protein were determined by fluorescence microscopy and/or Western blotting. As shown in this study, we were able to (i) clearly reduce the expression of EGFP alone and (ii) reduce expression of an EYFP fusion protein down to the level of the endogenous protein, both in a graded manner.     

Subcellular localization of proteins of Oryza sativa L. in the model tobacco and tomato plants

The cellular localization and molecular interactions are indicative of functions of a protein. The development of a simple and efficient method for subcellular localization of a protein is indispensable to elucidate gene function in plants. In this study, we assessed the feasibility of Agrobacterium-mediated transformation (agroinfiltration) of tobacco and tomato leaf tissue to follow intracellular targeting of proteins from rice fused to green fluorescent protein (GFP). For this, a simple in planta assay for subcellular localization of rice proteins in the heterologous host systems of tobacco and tomato leaf via transient transformation was developed. We have tested the applicability of this method by expressing GFP fusions of the putative antiphagocytic protein 1 (APP1) (OsAPP, LOC_Os03g56930) and ZOS3-18-C2H2 zinc-finger protein (OsZF1, LOC_Os03g55540) from Oryza sativa L. subsp. japonica in tobacco and tomato leaf tissues. Our results demonstrate the suitability of GFP as a reporter in gene expression studies in tomato cv. MicroTom. The use of GFP-fused proteins from rice for subcellular targeting in the heterologous hosts of tobacco and tomato plant systems has been confirmed.Key words: agroinfiltration, confocal microscopy, GFP fusion protein, tomato cv, microtom     

Green-fluorescent protein fusions for efficient characterization of nuclear targeting

The green-fluorescent protein (GFP) from Aequorea victoria has been shown to be a convenient and flexible reporter molecule within a variety of eukaryotic systems, including higher plants. It is particularly suited for applications in vivo, since the mechanism of fluorophore formation involves an intramolecular autoxidation and does not require exogenous co-factors. Unlike standard histochemical procedures of fixation and staining required for analysis of the cellular or tissue-specific expression of other popular reporter molecules, such as the β-glucuronidase (GUS) marker, analysis of GFP can be done in living cells with no specific pretreatments. This implies that GFP might also be particularly suited for studies of intracellular protein targeting. In this paper, the use of GUS is compared with that of GFP for the analysis of nuclear targeting in tobacco. A novel oligopeptide motif from a tobacco protein is described which confers nuclear localization of GUS. The use of this oligopeptide and two from potyviral proteins to target GFP to the nucleus is examined. An essential modification of GFP is described, which specifically increases its molecular weight to eliminate its passive penetration into the nucleus. Three examples of the targeting of these enlarged GFP molecules to the nucleus are illustrated. GFP, in combination with confocal microscopy, offers significant advantages over traditional methods of studying nuclear targeting.     

Understanding the folding of GFP using biophysical techniques

Green fluorescent protein (GFP) and its many variants are probably the most widely used proteins in medical and biological research, having been extensively engineered to act as markers of gene expression and protein localization, indicators of protein-protein interactions and biosensors. GFP first folds, before it can undergo an autocatalytic cyclization and oxidation reaction to form the chromophore, and in many applications the folding efficiency of GFP is known to limit its use. Here, we review the recent literature on protein engineering studies that have improved the folding properties of GFP. In addition, we discuss in detail the biophysical work on the folding of GFP that is beginning to reveal how this large and complex structure forms.     

Green fluorescent protein as a reporter for macromolecular localization in bacterial cells

Green fluorescent protein (GFP) is a highly useful fluorescent tag for studying the localization, structure, and dynamics of macromolecules in living cells, and has quickly become a primary tool for analysis of DNA and protein localization in prokaryotes. Several properties of GFP make it an attractive and versatile reporter. It is fluorescent and soluble in a wide variety of species, can be monitored noninvasively by external illumination, and needs no external substrates. Localization of GFP fusion proteins can be analyzed in live bacteria, therefore eliminating potential fixation artifacts and enabling real-time monitoring of dynamics in situ. Such real-time studies have been facilitated by brighter, more soluble GFP variants. In addition, red-shifted GFPs that can be excited by blue light have lessened the problem of UV-induced toxicity and photobleaching. The self-contained domain structure of GFP reduces the chance of major perturbations to GFP fluorescence by fused proteins and, conversely, to the activities of the proteins to which it is fused. As a result, many proteins fused to GFP retain their activities. The stability of GFP also allows detection of its fluorescence in vitro during protein purification and in cells fixed for indirect immunofluorescence and other staining protocols. Finally, the different properties of GFP variants have given rise to several technological innovations in the study of cellular physiology that should prove useful for studies in live bacteria. These include fluorescence resonance energy transfer (FRET) for studying protein-protein interactions and specially engineered GFP constructs for direct determination of cellular ion fluxes.     

Nuclear localization of enzymatically active green fluorescent protein-CTP:phosphocholine cytidylyltransferase alpha fusion protein is independent of cell cycle conditions and cell types

To address the recent controversy about the subcellular localization of CTP:phosphocholine cytidylyltransferase alpha (CTalpha), this study was designed to visualize green fluorescent protein (GFP). CTalpha fusion proteins directly and continuously under different conditions of cell cycling and in various cell lines. The GFP. CTalpha fusion proteins were enzymatically active and capable of rescuing mutant cells with a temperature-sensitive CT. The expressed GFP.CTalpha fusion protein was localized to the nucleus in all cell lines and required the N-terminal nuclear targeting sequence. Serum depletion/replenishment did not cause shuttling of CTalpha between the nucleus and cytoplasm. Moreover, the subcellular localization of CTalpha was examined continuously through all stages of the cell cycle in synchronized cells. No shuttling of CTalpha between the nucleus and cytoplasm was observed at any stage of the cell cycle. Stimulation of cells with oleate had no effect on the localization of CTalpha. The GFP.CTalpha lacking the nuclear targeting sequence stayed exclusively in the cytoplasm. Regardless of their localization, the GFP.CTalpha fusion proteins were equally active for phosphatidylcholine synthesis and mutant rescue. We conclude that the nuclear localization of CTalpha is a biological event independent of cell cycle in most mammalian cells and is unrelated to activation of phosphatidylcholine synthesis.     

Functional expression and cellular localization of a green fluorescent protein-tagged proline transporter in Aspergillus nidulans.

The PrnB protein is a highly specific proline transporter that belongs to an amino acid transporter family conserved in both prokaryotes and eukaryotes. In this work, we detected and analyzed the cellular localization of PrnB in vivo by means of green fluorescent protein (GFP) fusions. Several prnB-gfp gene fusions, driven by prnB native promoter sequences, were constructed and targeted to the genomic locus of a prnB null mutant. Chimeric proteins containing GFP fused to the C terminus of PrnB through a linker of two, four, or eight amino acids, with low potential to form secondary structure elements, were shown to be functional in vivo. A two-linker fusion results in partial complementation at both 25 and 37 degrees C. A four-linker fusion affords almost full complementation at 25 degrees C but partial complementation at 37 degrees C, whereas the eight-linker fusion results in partial complementation at both temperatures but in no GFP fluorescence. These results show that the number of linker amino acids is critical for the correct expression and/or translocation of PrnB-GFP fused proteins to the plasma membrane and for the fluorescence of the GFP. The expression of the four-linker PrnB-GFP transporter was detected and analyzed in vivo by both conventional fluorescence and confocal laser microscopy. This chimeric protein is localized in the plasma membrane, secondarily in large vacuoles found in the swollen conidial end of the germlings, and in other small intracellular structures observed as fluorescent granules. A strong correlation between known patterns of PrnB expression and intensity of PrnB-GFP fluorescence was observed. This work also demonstrates that the GFP fusion technology is a unique tool with which to study the expression and cellular localization of low-abundance transmembrane transporters expressed from their native promoters.     

Continuous translocation of Rac2 and the NADPH oxidase component p67(phox) during phagocytosis

In this study, the translocation of the NADPH oxidase components p67(phox) and Rac2 was studied during phagocytosis in living cells. For this purpose, green fluorescent protein (GFP)-tagged versions of these proteins were expressed in the myeloid cell line PLB-985. First, the correct localization of p67GFP and GFP-Rac2 was shown during phagocytosis of serum-treated zymosan by wild-type PLB-985 cells and PLB-985 X-CGD (chronic granulomatous disease) cells, which lack expression of flavocytochrome b(558). Subsequently, these constructs were used for fluorescence recovery after photobleaching studies to elucidate the turnover of these proteins on the phagosomal membrane. The turnover of p67GFP and GFP-Rac2 proved to be very high, indicating a continuous exchange of flavocytochrome b(558)-bound p67GFP and GFP-Rac2 for cytosolic, free p67GFP and GFP-Rac2. Furthermore, the importance of an intact actin cytoskeleton for correct localization of these proteins was investigated by disrupting the actin cytoskeleton with cytochalasin B. However, cytochalasin B treatment of PLB-985 cells did not alter the localization of p67GFP and GFP-Rac2 once phagocytosis was initiated. In addition, the continuous exchange of flavocytochrome b(558)-bound p67GFP and GFP-Rac2 for cytosolic p67GFP and GFP-Rac2 was still intact in cytochalasin B-treated cells, indicating that the translocation of these proteins does not depend on a rearrangement of the actin cytoskeleton.     

Nuclear translocation of Acinetobacter baumannii transposase induces DNA methylation of CpG regions in the promoters of E-cadherin gene

Nuclear targeting of bacterial proteins has emerged as a pathogenic mechanism whereby bacterial proteins induce host cell pathology. In this study, we examined nuclear targeting of Acinetobacter baumannii transposase (Tnp) and subsequent epigenetic changes in host cells. Tnp of A. baumannii ATCC 17978 possesses nuclear localization signals (NLSs), (225)RKRKRK(230). Transient expression of A. baumannii Tnp fused with green fluorescent protein (GFP) resulted in the nuclear localization of these proteins in COS-7 cells, whereas the truncated Tnp without NLSs fused with GFP were exclusively localized in the cytoplasm. A. baumannii Tnp was found in outer membrane vesicles, which delivered this protein to the nucleus of host cells. Nuclear expression of A. baumannii Tnp fused with GFP in A549 cells induced DNA methylation of CpG regions in the promoters of E-cadherin (CDH1) gene, whereas the cytoplasmic localization of the truncated Tnp without NLSs fused with GFP did not induce DNA methylation. DNA methylation in the promoters of E-cadherin gene induced by nuclear targeting of A. baumannii Tnp resulted in down-regulation of gene expression. In conclusion, our data show that nuclear traffic of A. baumannii Tnp induces DNA methylation of CpG regions in the promoters of E-cadherin gene, which subsequently down-regulates gene expression. This study provides a new insight into the epigenetic control of host genes by bacterial proteins.     

Investigation into the use of C- and N-terminal GFP fusion proteins for subcellular localization studies using reverse transfection microarrays

Reverse transfection microarrays were described recently as a high throughput method for studying gene function. We have investigated the use of this technology for determining the subcellular localization of proteins. Genes encoding 16 proteins with a variety of functions were placed in Gateway expression constructs with 3' or 5' green fluorescent protein (GFP) tags. These were then packaged in transfection reagent and spotted robotically onto a glass slide to form a reverse transfection array. HEK293T cells were grown over the surface of the array until confluent and GFP fluorescence visualized by confocal microscopy. All C-terminal fusion proteins localized to cellular compartments in accordance with previous studies and/or bioinformatic predictions. However, less than half of the N-terminal fusion proteins localized correctly. Of those that were not in concordance with the C-terminal tagged proteins, half did not exhibit expression and the remainder had differing subcellular localizations to the C-terminal fusion protein. This data indicates that N-terminal tagging with GFP adversely affects the protein localization in reverse transfection assays, whereas tagging with GFP at the C-terminal is generally better in preserving the localization of the native protein. We discuss these results in the context of developing high-throughput subcellular localization assays based on the reverse transfection array technology.     

植物细胞质介导GFP-NLS蛋白入核转运的HeLa细胞系统的建立

细胞核作为细胞中重要的遗传物质存储、复制和转录的结构,牵涉着大量信息和物质的传输活动,尤其是蛋白质的入核转运一直以来都是研究的热点问题之一。本文利用病毒SV40抗原蛋白中的核定位信号（nuclear localization signal,NLS）标记GFP蛋白,通过拟南芥细胞质的介导,利用HeLa细胞核建立起了研究蛋白质入核转运的半细胞体系。结果显示,植物细胞质结合NLS片段能改变GFP在HeLa细胞核内外的分布,实现对目标蛋白入核过程的介导,使GFP-NLS最后定位于细胞核内。这也意味着通过HeLa细胞建立起的半细胞体系能为蛋白入核转运研究提供一个有效的研究体系。