scholarly journals Investigation Into the use of C- and N-terminal GFP Fusion Proteins for Subcellular Localization Studies Using Reverse Transfection Microarrays

2004 ◽  
Vol 5 (4) ◽  
pp. 342-353 ◽  
Author(s):  
Ella Palmer ◽  
Tom Freeman
Keyword(s):  
Subcellular Localization ◽  
High Throughput ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Genes Encoding ◽  
Reverse Transfection ◽  
C And N ◽  
Green Fluorescent ◽  
Cellular Compartments

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.

scholarly journals pFPL Vectors for High-Throughput Protein Localization in Fungi: Detecting Cytoplasmic Accumulation of Putative Effector Proteins

2015 ◽  
Vol 28 (2) ◽  
pp. 107-121 ◽  
Author(s):  
Xiaoyan Gong ◽  
Oscar Hurtado ◽  
Baohua Wang ◽  
Congqing Wu ◽  
Mihwa Yi ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Protein Localization ◽  
Yellow Fluorescent Protein ◽  
Effector Proteins ◽  
Fungal Transformation ◽  
In Planta ◽  
Green Fluorescent

As part of a large-scale project whose goal was to identify candidate effector proteins in Magnaporthe oryzae, we developed a suite of vectors that facilitate high-throughput protein localization experiments in fungi. These vectors utilize Gateway recombinational cloning to place a gene's promoter and coding sequences upstream and in frame with enhanced cyan fluorescent protein, green fluorescent protein (GFP), monomeric red fluorescence protein (mRFP), and yellow fluorescent protein or a nucleus-targeted mCHERRY variant. The respective Gateway cassettes were incorporated into Agrobacterium-based plasmids to allow efficient fungal transformation using hygromycin or geneticin resistance selection. mRFP proved to be more sensitive than the GFP spectral variants for monitoring proteins secreted in planta; and extensive testing showed that Gateway-derived fusion proteins produced localization patterns identical to their “directly fused” counterparts. Use of plasmid for fungal protein localization (pFPL) vectors with two different selectable markers provided a convenient way to label fungal cells with different fluorescent proteins. We demonstrate the utility of the pFPL vectors for identifying candidate effector proteins and we highlight a number of important factors that must be taken into consideration when screening for proteins that are translocated across the host plasma membrane.

scholarly journals Dynamic Patterns of Subcellular Protein Localization during Spore Coat Morphogenesis in Bacillus subtilis

2004 ◽  
Vol 186 (14) ◽  
pp. 4441-4448 ◽  
Author(s):  
Christiaan van Ooij ◽  
Patrick Eichenberger ◽  
Richard Losick
Keyword(s):  
Bacillus Subtilis ◽  
Subcellular Localization ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Distinct Pattern ◽  
Spore Coat ◽  
Short Axis ◽  
Subcellular Protein Localization ◽  
Associated Proteins ◽  
Green Fluorescent

ABSTRACT Endospores of Bacillus subtilis are encased in a thick, proteinaceous shell known as the coat, which is composed of a large number of different proteins. Here we report the identification of three previously uncharacterized coat-associated proteins, YabP, YheD, and YutH, and their patterns of subcellular localization during the process of sporulation, obtained by using fusions of the proteins to the green fluorescent protein (GFP). YabP-GFP was found to form both a shell and a ring around the center of the forespore across the short axis of the sporangium. YheD-GFP, in contrast, formed two rings around the forespore that were offset from its midpoint, before it eventually redistributed to form a shell around the developing spore. Finally, YutH-GFP initially localized to a focus at one end of the forespore, which then underwent transformation into a ring that was located adjacent to the forespore. Next, the ring became a cap at the mother cell pole of the forespore that eventually spread around the entire developing spore. Thus, each protein exhibited its own distinct pattern of subcellular localization during the course of coat morphogenesis. We concluded that spore coat assembly is a dynamic process involving diverse patterns of protein assembly and localization.

scholarly journals Functional Expression and Subcellular Localization of the Aflatoxin Pathway Enzyme Ver-1 Fused to Enhanced Green Fluorescent Protein

2008 ◽  
Vol 74 (20) ◽  
pp. 6385-6396 ◽  
Author(s):  
Sung-Yong Hong ◽  
John E. Linz
Keyword(s):  
Green Fluorescent Protein ◽  
Subcellular Localization ◽  
Fusion Proteins ◽  
Laser Scanning ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Substrate Surface ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Green Fluorescent

ABSTRACT Aflatoxin, a mycotoxin synthesized by Aspergillus spp., is among the most potent naturally occurring carcinogens known. Little is known about the subcellular organization of aflatoxin synthesis. Previously, we used transmission electron microscopy and immunogold labeling to demonstrate that the late aflatoxin enzyme OmtA localizes primarily to vacuoles in fungal cells on the substrate surface of colonies. In the present work, we monitored subcellular localization of Ver-1 in real time in living cells. Aspergillus parasiticus strain CS10-N2 was transformed with plasmid constructs that express enhanced green fluorescent protein (EGFP) fused to Ver-1. Analysis of transformants demonstrated that EGFP fused to Ver-1 at either the N or C terminus functionally complemented nonfunctional Ver-1 in recipient cells. Western blot analysis detected predominantly full-length Ver-1 fusion proteins in transformants. Confocal laser scanning microscopy demonstrated that Ver-1 fusion proteins localized in the cytoplasm and in the lumen of up to 80% of the vacuoles in hyphae grown for 48 h on solid media. Control EGFP (no Ver-1) expressed in transformants was observed in only 13% of the vacuoles at this time. These data support a model in which middle and late aflatoxin enzymes are synthesized in the cytoplasm and transported to vacuoles, where they participate in aflatoxin synthesis.

scholarly journals A Photostable Green Fluorescent Protein Variant for Analysis of Protein Localization in Candida albicans

2009 ◽  
Vol 9 (1) ◽  
pp. 224-226 ◽  
Author(s):  
Chengda Zhang ◽  
James B. Konopka
Keyword(s):  
Candida Albicans ◽  
Green Fluorescent Protein ◽  
Codon Usage ◽  
Signal Intensity ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Protein Variant ◽  
Green Fluorescent ◽  
Green Fluorescent Protein Variant

ABSTRACT Fusions to the green fluorescent protein (GFP) are an effective way to monitor protein localization. However, altered codon usage in Candida species has delayed implementation of new variants. Examination of three new GFP variants in Candida albicans showed that one has higher signal intensity and increased resistance to photobleaching.

scholarly journals Neutralization Assay Using a Modified Vaccinia Virus Ankara Vector Expressing the Green Fluorescent Protein Is a High-Throughput Method To Monitor the Humoral Immune Response against Vaccinia Virus

2004 ◽  
Vol 11 (2) ◽  
pp. 406-410 ◽  
Author(s):  
Antonio Cosma ◽  
Silja Bühler ◽  
Rashmi Nagaraj ◽  
Caroline Staib ◽  
Anna-Lena Hammarin ◽  
...  
Keyword(s):  
Immune Response ◽  
Green Fluorescent Protein ◽  
Vaccinia Virus ◽  
High Throughput ◽  
Neutralizing Antibodies ◽  
Fluorescent Protein ◽  
Safety Concern ◽  
Neutralization Assay ◽  
Modified Vaccinia Virus Ankara ◽  
Green Fluorescent

ABSTRACT Vaccination against smallpox is again considered in order to face a possible bioterrorist threat, but the nature and the level of the immune response needed to protect a person from smallpox after vaccination are not totally understood. Therefore, simple, rapid, and accurate assays to evaluate the immune response to vaccinia virus need to be developed. Neutralization assays are usually considered good predictors of vaccine efficacy and more informative with regard to protection than binding assays. Currently, the presence of neutralizing antibodies to vaccinia virus is measured using a plaque reduction neutralization test, but this method is time-consuming and labor-intensive and has a subjective readout. Here, we describe an innovative neutralization assay based on a modified vaccinia virus Ankara (MVA) vector expressing the green fluorescent protein (MVA-gfp). This MVA-gfp neutralization assay is rapid and sensitive and has a high-throughput potential. Thus, it is suitable to monitor the immune response and eventually the efficacy of a large campaign of vaccination against smallpox and to study the vector-specific immune response in clinical trials that use genetically engineered vaccinia viruses. Most importantly, application of the highly attenuated MVA eliminates the safety concern in using the replication-competent vaccinia virus in the standard clinical laboratory.

scholarly journals Fluorescence-Based Reporter for Gauging Cyclic Di-GMP Levels in Pseudomonas aeruginosa

2012 ◽  
Vol 78 (15) ◽  
pp. 5060-5069 ◽  
Author(s):  
Morten T. Rybtke ◽  
Bradley R. Borlee ◽  
Keiji Murakami ◽  
Yasuhiko Irie ◽  
Morten Hentzer ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Fluorescent Protein ◽  
Subsequent Development ◽  
Cellular Level ◽  
Host Immune System ◽  
Chronic Infections ◽  
Content Type ◽  
Genes Encoding ◽  
Green Fluorescent

ABSTRACTThe increased tolerance toward the host immune system and antibiotics displayed by biofilm-formingPseudomonas aeruginosaand other bacteria in chronic infections such as cystic fibrosis bronchopneumonia is of major concern. Targeting of biofilm formation is believed to be a key aspect in the development of novel antipathogenic drugs that can augment the effect of classic antibiotics by decreasing antimicrobial tolerance. The second messenger cyclic di-GMP is a positive regulator of biofilm formation, and cyclic di-GMP signaling is now regarded as a potential target for the development of antipathogenic compounds. Here we describe the development of fluorescent monitors that can gauge the cellular level of cyclic di-GMP inP. aeruginosa. We have created cyclic di-GMP level reporters by transcriptionally fusing the cyclic di-GMP-responsivecdrApromoter to genes encoding green fluorescent protein. We show that the reporter constructs give a fluorescent readout of the intracellular level of cyclic di-GMP inP. aeruginosastrains with different levels of cyclic di-GMP. Furthermore, we show that the reporters are able to detect increased turnover of cyclic di-GMP mediated by treatment ofP. aeruginosawith the phosphodiesterase inducer nitric oxide. Considering that biofilm formation is a necessity for the subsequent development of a chronic infection and therefore a pathogenicity trait, the reporters display a significant potential for use in the identification of novel antipathogenic compounds targeting cyclic di-GMP signaling, as well as for use in research aiming at understanding the biofilm biology ofP. aeruginosa.

scholarly journals Recombinant Lassa Virus Expressing Green Fluorescent Protein as a Tool for High-Throughput Drug Screens and Neutralizing Antibody Assays

Viruses ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 655 ◽  
Author(s):  
Yíngyún Caì ◽  
Masaharu Iwasaki ◽  
Brett Beitzel ◽  
Shuīqìng Yú ◽  
Elena Postnikova ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
High Throughput ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Neutralizing Antibody ◽  
Quantitative Detection ◽  
Lassa Virus ◽  
Wild Type ◽  
Green Fluorescent

Lassa virus (LASV), a mammarenavirus, infects an estimated 100,000–300,000 individuals yearly in western Africa and frequently causes lethal disease. Currently, no LASV-specific antivirals or vaccines are commercially available for prevention or treatment of Lassa fever, the disease caused by LASV. The development of medical countermeasure screening platforms is a crucial step to yield licensable products. Using reverse genetics, we generated a recombinant wild-type LASV (rLASV-WT) and a modified version thereof encoding a cleavable green fluorescent protein (GFP) as a reporter for rapid and quantitative detection of infection (rLASV-GFP). Both rLASV-WT and wild-type LASV exhibited similar growth kinetics in cultured cells, whereas growth of rLASV-GFP was slightly impaired. GFP reporter expression by rLASV-GFP remained stable over several serial passages in Vero cells. Using two well-characterized broad-spectrum antivirals known to inhibit LASV infection, favipiravir and ribavirin, we demonstrate that rLASV-GFP is a suitable screening tool for the identification of LASV infection inhibitors. Building on these findings, we established a rLASV-GFP-based high-throughput drug discovery screen and an rLASV-GFP-based antibody neutralization assay. Both platforms, now available as a standard tool at the IRF-Frederick (an international resource), will accelerate anti-LASV medical countermeasure discovery and reduce costs of antiviral screens in maximum containment laboratories.

scholarly journals Multiple Adhesin-Like Functions of Xanthomonas oryzae pv. oryzae Are Involved in Promoting Leaf Attachment, Entry, and Virulence on Rice

2009 ◽  
Vol 22 (1) ◽  
pp. 73-85 ◽  
Author(s):  
Amit Das ◽  
Nandini Rangaraj ◽  
Ramesh V. Sonti
Keyword(s):  
Bacterial Adhesion ◽  
Bacterial Blight ◽  
Fluorescent Protein ◽  
Protein A ◽  
Disease Process ◽  
Xanthomonas Oryzae ◽  
Type Iv ◽  
Genes Encoding ◽  
Green Fluorescent

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial blight of rice. We have used enhanced green fluorescent protein-tagged X. oryzae pv. oryzae cells in conjunction with confocal microscopy to monitor the role of several adhesin-like functions in bacterial adhesion to leaf surface and early stages of leaf entry. Mutations in genes encoding either the Xanthomonas adhesin-like protein A (XadA) or its paralog, Xanthomonas adhesin-like protein B (XadB), as well as the X. oryzae pv. oryzae homolog of Yersinia autotransporter-like protein H (YapH), exhibit deficiencies in leaf attachment or entry. A mutation in the X. oryzae pv. oryzae pilQ gene, which is predicted to encode the type IV pilus secretin, appears to have no effect on leaf attachment or entry. The xadA– mutant is deficient in the ability to cause disease following surface inoculation while the XadB, YapH, and PilQ functions are less important than XadA for this process. The xadA– and xadB– mutants have no effect on virulence following wound inoculation whereas the yapH– and pilQ– mutants are always virulence deficient following wound inoculation. Overall, these results indicate that multiple adhesin-like functions are involved in promoting virulence of X. oryzae pv. oryzae, with preferential involvement of individual functions at different stages of the disease process.

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