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https://www.readbyqxmd.com/read/29621251/ultrastructural-localisation-of-protein-interactions-using-conditionally-stable-nanobodies
#1
Nicholas Ariotti, James Rae, Nichole Giles, Nick Martel, Emma Sierecki, Yann Gambin, Thomas E Hall, Robert G Parton
We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome...
April 5, 2018: PLoS Biology
https://www.readbyqxmd.com/read/29451720/detection-of-membrane-protein-protein-interaction-in-planta-based-on-dual-intein-coupled-tripartite-split-gfp-association
#2
Tzu-Yin Liu, Wen-Chun Chou, Wei-Yuan Chen, Ching-Yi Chu, Chen-Yi Dai, Pei-Yu Wu
Despite a great interest of identification of protein-protein interactions (PPIs) in biological systems, only few attempts have been made for a large-scale PPI screen in planta. Unlike biochemical assays, bimolecular fluorescence complementation (BiFC) allows visualization of the transient and weak PPIs in vivo at the subcellular resolution. However, when the non-fluorescent fragments are highly expressed, the spontaneous and irreversible self-assembly of the split halves can easily generate false positives...
February 16, 2018: Plant Journal: for Cell and Molecular Biology
https://www.readbyqxmd.com/read/29436229/transpeptidation-mediated-assembly-of-tripartite-split-gfp-for-label-free-assay-of-sortase-activity
#3
Juan Zhang, Menglin Wang, Rui Tang, Yanan Liu, Chunyang Lei, Yan Huang, Zhou Nie, Shouzhuo Yao
Transpeptidation of surface proteins catalyzed by the transpeptidase sortase plays a critical role in the infection process of gram-positive pathogen, and probing sortase activity and screening its inhibitors are of great significance to fundamental biological research and pharmaceutical development, especially novel anti-virulence drug design. Herein, we developed a novel fluorescent biosensor to detect sortase activity based on a transpeptidation-triggered assembly of tripartite split green fluorescent protein (split GFP)...
February 13, 2018: Analytical Chemistry
https://www.readbyqxmd.com/read/29390129/direct-visualization-of-the-expression-and-localization-of-chlamydial-effector-proteins-within-infected-host-cells
#4
Xiaogang Wang, Kevin Hybiske, Richard S Stephens
Chlamydia secrete into host cells a diverse array of effector proteins, but progress in characterizing the spatiotemporal localization of these proteins has been hindered by a paucity of genetic approaches in Chlamydia and also by the challenge of studying these proteins within the live cellular environment. We adapted a split-Green Fluorescent Protein (GFP) system for use in Chlamydia to label chlamydial effector proteins and track their localization in host cells under native environment. The efficacy of this system was demonstrated by detecting several known Chlamydia proteins including IncA, CT005, and CT694...
January 30, 2018: Pathogens and Disease
https://www.readbyqxmd.com/read/29322444/constructing-gfp-based-reporter-to-study-back-splicing-and-translation-of-circular-rna
#5
Yun Yang, Zefeng Wang
Human transcriptome contains a large number of circular RNAs (circRNAs) that are mainly produced by back splicing of pre-mRNA. Here we describe a minigene reporter system containing a single exon encoding split GFP in reverse order, which can be efficiently back spliced to produce a circRNA encoding intact GFP gene. This simple reporter system can be adopted to study how different cis-elements and trans-factors affect circRNA production, and also can serve as a reliable system to measure the activity of IRES-mediated translation...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29232933/a-split-luciferase-reporter-recognizing-gfp-and-mcherry-tags-to-facilitate-studies-of-protein-protein-interactions
#6
Mehdi Moustaqil, Akshay Bhumkar, Laura Gonzalez, Lisa Raoul, Dominic J B Hunter, Pascal Carrive, Emma Sierecki, Yann Gambin
The use of fluorescently-tagged proteins in microscopy has become routine, and anti-GFP (Green fluorescent protein) affinity matrices are increasingly used in proteomics protocols. However, some protein-protein interactions assays, such as protein complementation assays (PCA), require recloning of each protein as a fusion with the different parts of the complementation system. Here we describe a generic system where the complementation is separated from the proteins and can be directly used with fluorescently-tagged proteins...
December 11, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/29229997/splics-a-split-green-fluorescent-protein-based-contact-site-sensor-for-narrow-and-wide-heterotypic-organelle-juxtaposition
#7
Domenico Cieri, Mattia Vicario, Marta Giacomello, Francesca Vallese, Riccardo Filadi, Tina Wagner, Tullio Pozzan, Paola Pizzo, Luca Scorrano, Marisa Brini, Tito Calì
Contact sites are discrete areas of organelle proximity that coordinate essential physiological processes across membranes, including Ca2+ signaling, lipid biosynthesis, apoptosis, and autophagy. However, tools to easily image inter-organelle proximity over a range of distances in living cells and in vivo are lacking. Here we report a split-GFP-based contact site sensor (SPLICS) engineered to fluoresce when organelles are in proximity. Two SPLICS versions efficiently measured narrow (8-10 nm) and wide (40-50 nm) juxtapositions between endoplasmic reticulum and mitochondria, documenting the existence of at least two types of contact sites in human cells...
December 11, 2017: Cell Death and Differentiation
https://www.readbyqxmd.com/read/29193968/structural-insight-into-the-photochemistry-of-split-green-fluorescent-proteins-a-unique-role-for-a-his-tag
#8
Alan Deng, Steven G Boxer
Oligohistidine affinity tags (His-tags) are commonly fused to proteins to aid in their purification via metal affinity chromatography. These His-tags are generally assumed to have minimal impact on the properties of the fusion protein, as they have no propensity to form ordered elements, and are small enough not to significantly affect the solubility or size. Here we report structures of two variants of truncated green fluorescent protein (GFP), i.e., split GFP with a β-strand removed, that were found to behave differently in the presence of light...
January 10, 2018: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/29192060/high-content-tripartite-split-gfp-cell-based-assays-to-screen-for-modulators-of-small-gtpase-activation
#9
Faten Koraïchi, Rémi Gence, Catherine Bouchenot, Sarah Grosjean, Isabelle Lajoie-Mazenc, Gilles Favre, Stéphanie Cabantous
The human Ras superfamily of small GTPases controls essential cellular processes such as gene expression and cell proliferation. As their deregulation is widely associated with human cancer, small GTPases and their regulatory proteins have become increasingly attractive for the development of novel therapeutics. Classical methods to monitor GTPase activation include pulldown assays that limit the analysis of GTP-bound form of proteins from cell lysates. Alternatively, live-cell FRET biosensors may be used to study GTPase activation dynamics in response to stimuli, but these sensors often require further optimization for high-throughput applications...
January 8, 2018: Journal of Cell Science
https://www.readbyqxmd.com/read/29158224/a-novel-fluorescent-reporter-detects-plastic-remodeling-of-mitochondria-er-contact-sites
#10
Zhaoying Yang, Xiaocui Zhao, Jiashen Xu, Weina Shang, Chao Tong
Mitochondria-ER contact sites (MERCs) enable communication between the ER and mitochondria and serve as platforms for many cellular events, including autophagy. Nonetheless, the molecular organization of MERCs is not known, and there is no bona fide marker of these contact sites in mammalian cells. In this study, we designed a genetically encoded reporter using split GFP protein for labeling MERCs. We subsequently analyzed its distribution and dynamics during the cell cycle and under stressful cellular conditions such as starvation, apoptosis and ER stress...
January 4, 2018: Journal of Cell Science
https://www.readbyqxmd.com/read/29154784/tgf-%C3%AE-1-signaling-regulates-mouse-hepatic-stellate-cell-differentiation-via-the-jagged1-notch-pathway
#11
Yasen Aimaiti, Xin Jin, Wei Wang, Zixin Chen, Dewei Li
AIMS: We tested whether transforming growth factor β1 (TGF-β1) signaling plays an important role in hepatic stellate cell differentiation fate and investigated the role of Jagged1/Notch in this process. MATERIALS AND METHODS: TGF-β1 was overexpressed and transforming growth factor receptor 1 (TGF-β-R1) was knocked down by a lentiviral vector in mouse hepatic stellate cells (mHSCs). Transfection efficiency was assessed with immunofluorescence, quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and western blotting...
January 1, 2018: Life Sciences
https://www.readbyqxmd.com/read/29108650/biophysical-characterization-of-genetically-encoded-voltage-sensor-asap1-dynamic-range-improvement
#12
Elizabeth E L Lee, Francisco Bezanilla
Recent work has introduced a new fluorescent voltage sensor, ASAP1, which can monitor rapid trains of action potentials in cultured neurons. This indicator is based on the Gallus gallus voltage-sensitive phosphatase with the phosphatase domain removed and a circularly permuted GFP placed in the S3-S4 linker. However, many of the biophysical details of this indicator remain unknown. In this work, we study the biophysical properties of ASAP1. Using the cut-open voltage clamp technique, we have simultaneously recorded fluorescence signals and gating currents from Xenopus laevis oocytes expressing ASAP1...
November 21, 2017: Biophysical Journal
https://www.readbyqxmd.com/read/29097689/marked-bias-towards-spontaneous-synaptic-inhibition-distinguishes-non-adapting-from-adapting-layer-5-pyramidal-neurons-in-the-barrel-cortex
#13
Ion R Popescu, Kathy Q Le, Rocío Palenzuela, Rebecca Voglewede, Ricardo Mostany
Pyramidal neuron subtypes differ in intrinsic electrophysiology properties and dendritic morphology. However, do different pyramidal neuron subtypes also receive synaptic inputs that are dissimilar in frequency and in excitation/inhibition balance? Unsupervised clustering of three intrinsic parameters that vary by cell subtype - the slow afterhyperpolarization, the sag, and the spike frequency adaptation - split layer 5 barrel cortex pyramidal neurons into two clusters: one of adapting cells and one of non-adapting cells, corresponding to previously described thin- and thick-tufted pyramidal neurons, respectively...
November 2, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29070802/split-gfp-technologies-to-structurally-characterize-and-quantify-functional-biomolecular-interactions-of-ftd-related-proteins
#14
Chiara Foglieni, Stéphanie Papin, Agnese Salvadè, Tariq Afroz, Sandra Pinton, Giona Pedrioli, Giorgio Ulrich, Magdalini Polymenidou, Paolo Paganetti
Protein multimerization in physiological and pathological conditions constitutes an intrinsic trait of proteins related to neurodegeneration. Recent evidence shows that TDP-43, a RNA-binding protein associated with frontotemporal dementia and amyotrophic lateral sclerosis, exists in a physiological and functional nuclear oligomeric form, whose destabilization may represent a prerequisite for misfolding, toxicity and subsequent pathological deposition. Here we show the parallel implementation of two split GFP technologies, the GFP bimolecular and trimolecular fluorescence complementation (biFC and triFC) in the context of TDP-43 self-assembly...
October 25, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29069446/6-4-pp-photolyase-encoded-by-atuvr3-is-localized-in-nuclei-chloroplasts-and-mitochondria-and-its-expression-is-down-regulated-by-light-in-a-photosynthesis-dependent-manner
#15
Agnieszka Katarzyna Banas, Pawel Hermanowicz, Olga Sztatelman, Justyna Labuz, Chhavi Aggarwal, Piotr Zglobicki, Dominika Jagiello-Flasinska, Wojciech Strzalka
Pyrimidine dimers are the most important DNA lesions induced by UVB irradiation. They can be repaired directly by photoreactivation or indirectly by the excision repair pathways. Photoreactivation is performed by photolyases, enzymes which bind to the dimers and use the energy of blue light or UVA to split bonds between adjacent pyrimidines. Arabidopsis thaliana has three known photolyases: AtPHR1, AtCRY3 and AtUVR3. Little is known about the cellular localization and regulation of AtUVR3 expression. We have found that its transcript level is down-regulated by light (red, blue or white) in a photosynthesis-dependent manner...
October 24, 2017: Plant & Cell Physiology
https://www.readbyqxmd.com/read/28991414/in-situ-spatial-complementation-of-aptamer-mediated-recognition-enables-live-cell-imaging-of-native-rna-transcripts-in-real-time
#16
Zejun Wang, Yao Luo, Xiaodong Xie, Xingjie Hu, Haiyun Song, Yun Zhao, Jiye Shi, Lihua Wang, Gennadi Glinsky, Nan Chen, Ratnesh Lal, Chunhai Fan
Direct cellular imaging of the localization and dynamics of biomolecules helps to understand their function and reveals novel mechanisms at the single-cell resolution. In contrast to routine fluorescent-protein-based protein imaging, technology for RNA imaging remains less well explored because of the lack of enabling technology. Herein, we report the development of an aptamer-initiated fluorescence complementation (AiFC) method for RNA imaging by engineering a green fluorescence protein (GFP)-mimicking turn-on RNA aptamer, Broccoli, into two split fragments that could tandemly bind to target mRNA...
January 22, 2018: Angewandte Chemie
https://www.readbyqxmd.com/read/28977453/enrichment-of-dynamic-chromosomal-crosslinks-drive-phase-separation-of-the-nucleolus
#17
Caitlin Hult, David Adalsteinsson, Paula A Vasquez, Josh Lawrimore, Maggie Bennett, Alyssa York, Diana Cook, Elaine Yeh, Mark Gregory Forest, Kerry Bloom
Regions of highly repetitive DNA, such as those found in the nucleolus, show a self-organization that is marked by spatial segregation and frequent self-interaction. The mechanisms that underlie the sequestration of these sub-domains are largely unknown. Using a stochastic, bead-spring representation of chromatin in budding yeast, we find enrichment of protein-mediated, dynamic chromosomal cross-links recapitulates the segregation, morphology and self-interaction of the nucleolus. Rates and enrichment of dynamic crosslinking have profound consequences on domain morphology...
November 2, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28956769/epstein-barr-virus-fusion-with-epithelial-cells-triggered-by-gb-is-restricted-by-a-gl-glycosylation-site
#18
Britta S Möhl, Jia Chen, Seo Jin Park, Theodore S Jardetzky, Richard Longnecker
Epstein-Barr virus (EBV) entry into epithelial cells is mediated by the conserved core fusion machinery, composed of the fusogen gB and the receptor-binding complex gH/gL. The heterodimeric gH/gL complex binds to the EBV epithelial cell receptor or gp42, which binds to the B-cell receptor, triggering gB-mediated fusion of the virion envelope with cellular membranes. Our previous study found that the gL glycosylation mutant N69L/S71V had an epithelial cell-specific hyperfusogenic phenotype. To study the influence of this gL mutant on the initiation and kinetics of gB-driven epithelial cell fusion, we established a virus-free split-green fluorescent protein cell-cell fusion assay that enables real-time measurements of membrane fusion using live cells...
December 1, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28945234/a-homodimer-interface-without-base-pairs-in-an-rna-mimic-of-red-fluorescent-protein
#19
Katherine Deigan Warner, Ljiljana Sjekloća, Wenjiao Song, Grigory S Filonov, Samie R Jaffrey, Adrian R Ferré-D'Amaré
Corn, a 28-nucleotide RNA, increases yellow fluorescence of its cognate ligand 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO) by >400-fold. Corn was selected in vitro to overcome limitations of other fluorogenic RNAs, particularly rapid photobleaching. We now report the Corn-DFHO co-crystal structure, discovering that the functional species is a quasisymmetric homodimer. Unusually, the dimer interface, in which six unpaired adenosines break overall two-fold symmetry, lacks any intermolecular base pairs...
November 2017: Nature Chemical Biology
https://www.readbyqxmd.com/read/28842906/application-of-split-gfp-reassembly-assay-to-study-myogenesis-and-myofusion-in-vitro
#20
Manami Kodaka, Xiaoyin Xu, Zeyu Yang, Junichi Maruyama, Yutaka Hata
Green fluorescent protein (GFP) is composed of 11 β-strands, and loses GFP signals, when divided into the N-terminal ten β-strands (GFP1-10) and the C-terminal last β-strand (GFP11). However, when GFP1-10 and GFP11 encounter, they reassemble into the fluorescent GFP. We expressed GFP1-10 and blasticidin resistance gene product-fused GFP11 (BSR-GFP11) in C2C12 cells. Both the cell lines do not show GFP but when they undergo myogenesis and myofusion, GFP1-10 and BSR-GFP11 form the fluorescent complex in multi-nuclear myotubes, so that GFP signals reflect myogenesis and myofusion...
2017: Methods in Molecular Biology
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