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https://www.readbyqxmd.com/read/28842906/application-of-split-gfp-reassembly-assay-to-study-myogenesis-and-myofusion-in-vitro
#1
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
https://www.readbyqxmd.com/read/28767038/microtubule-dependent-ribosome-localization-in-c-elegans-neurons
#2
Kentaro Noma, Alexandr Goncharov, Mark H Ellisman, Yishi Jin
Subcellular localization of ribosomes defines the location and capacity for protein synthesis. Methods for in vivo visualizing ribosomes in multicellular organisms are desirable in mechanistic investigations of the cell biology of ribosome dynamics. Here, we developed an approach using split GFP for tissue-specific visualization of ribosomes in Caenorhabditis elegans. Labeled ribosomes are detected as fluorescent puncta in the axons and synaptic terminals of specific neuron types, correlating with ribosome distribution at the ultrastructural level...
August 2, 2017: ELife
https://www.readbyqxmd.com/read/28621789/display-of-functional-proteins-on-supramolecular-peptide-nanofibrils-using-a-split-protein-strategy
#3
John T M DiMaio, Danielle M Raymond, Bradley L Nilsson
The display of functional proteins on self-assembled peptide nanofibrils is challenging since the steric bulk of proteins attached to simple self-assembling peptides often impedes incorporation into nanofibrils. Herein is described a split-protein strategy to tether functional proteins to preassembled peptide nanofibrils. In this strategy, a short affinity motif peptide derived from a split protein system is appended to a self-assembly motif (the amphipathic Ac-(FKFE)2-NH2 peptide) to form an affinity-assembly fusion peptide...
June 27, 2017: Organic & Biomolecular Chemistry
https://www.readbyqxmd.com/read/28619883/spatiotemporal-monitoring-of-pseudomonas-syringae-effectors-via-type-iii-secretion-using-split-fluorescent-protein-fragments
#4
Eunsook Park, Hye-Young Lee, Jongchan Woo, Doil Choi, Savithramma P Dinesh-Kumar
Pathogenic gram-negative bacteria cause serious diseases in animals and plants. These bacterial pathogens use the type III secretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellular compartments to attenuate immune responses by altering biological processes of the host cells. The fluorescent protein (FP)-based approach to monitor effectors secreted from bacteria into the host cells is not possible because the folded FP prevents effector delivery through the T3SS Therefore, we optimized an improved variant of self-assembling split super-folder green fluorescent protein (sfGFP(OPT)) system to investigate the spatiotemporal dynamics of effectors delivered through bacterial T3SS into plant cells...
July 2017: Plant Cell
https://www.readbyqxmd.com/read/28619616/activity-independent-screening-of-secreted-proteins-using-split-gfp
#5
Andreas Knapp, Myriam Ripphahn, Kristina Volkenborn, Pia Skoczinski, Karl-Erich Jaeger
The large-scale industrial production of proteins requires efficient secretion, as provided, for instance, by the Sec system of Gram-positive bacteria. Protein engineering approaches to optimize secretion often involve the screening of large libraries, e.g. comprising a target protein fused to many different signal peptides. Respective high-throughput screening methods are usually based on photometric or fluorimetric assays enabling fast and simple determination of enzymatic activities. Here, we report on an alternative method for quantification of secreted proteins based on the split GFP assay...
June 12, 2017: Journal of Biotechnology
https://www.readbyqxmd.com/read/28551989/optimized-fluorescence-complementation-platform-for-visualizing-salmonella-effector-proteins-reveals-distinctly-different-intracellular-niches-in-different-cell-types
#6
Alexandra M Young, Michael Minson, Sarah E McQuate, Amy E Palmer
The bacterial pathogen Salmonella uses sophisticated type III secretion systems (T3SS) to translocate and deliver bacterial effector proteins into host cells to establish infection. Monitoring these important virulence determinants in the context of live infections is a key step in defining the dynamic interface between the host and pathogen. Here, we provide a modular labeling platform based on fluorescence complementation with split-GFP that permits facile tagging of new Salmonella effector proteins. We demonstrate enhancement of split-GFP complementation signals by manipulating the promoter or by multimerizing the fluorescent tag and visualize three effector proteins, SseF, SseG, and SlrP, that have never before been visualized over time during infection of live cells...
June 9, 2017: ACS Infectious Diseases
https://www.readbyqxmd.com/read/28470605/split-gfp-complementation-as-reporter-of-membrane-protein-expression-and-stability-in-e-coli-a-tool-to-engineer-stability-in-a-lat-transporter
#7
Ekaitz Errasti-Murugarren, Arturo Rodríguez-Banqueri, José Luis Vázquez-Ibar
Obtaining enough quantity of recombinant membrane transport proteins with optimal purity and stability for structural studies is a remarkable challenge. In this chapter, we describe a protocol to engineer SteT, the amino acid transporter of Bacillus subtilis, in order to improve its heterologous expression in Escherichia coli and its stability in detergent micelles. We built a library of 70 SteT mutants, combining a random mutagenesis protocol with a split GFP assay as reporter of protein folding and membrane insertion...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28391244/introducing-inducible-fluorescent-split-cholesterol-oxidase-to-mammalian-cells
#8
Konstantin G Chernov, Maarit Neuvonen, Ivonne Brock, Elina Ikonen, Vladislav V Verkhusha
Cholesterol oxidase (COase) is a bacterial enzyme catalyzing the first step in the biodegradation of cholesterol. COase is an important biotechnological tool for clinical diagnostics and production of steroid drugs and insecticides. It is also used for tracking intracellular cholesterol; however, its utility is limited by the lack of an efficient temporal control of its activity. To overcome this we have developed a regulatable fragment complementation system for COase cloned from Chromobacterium sp. The enzyme was split into two moieties that were fused to FKBP (FK506-binding protein) and FRB (rapamycin-binding domain) pair and split GFP fragments...
May 26, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28358924/processing-and-targeting-of-proteins-derived-from-polyprotein-with-2a-and-lp4-2a-as-peptide-linkers-in-a-maize-expression-system
#9
He Sun, Ni Zhou, Hai Wang, Dafang Huang, Zhihong Lang
In the transformation of multiple genes, gene fusion is an attractive alternative to other methods, including sexual crossing, re-transformation, and co-transformation, among others. The 2A peptide from the foot-and-mouth disease virus (FMDV) causes the co-translational "cleavage" of polyprotein and operates in a wide variety of eukaryotic cells. LP4, a linker peptide that originates from a natural polyprotein occurring in the seed of Impatiens balsamina, can be split between the first and second amino acids in post-translational processing...
2017: PloS One
https://www.readbyqxmd.com/read/28242710/mechanism-and-bottlenecks-in-strand-photodissociation-of-split-green-fluorescent-proteins-gfps
#10
Chi-Yun Lin, Johan Both, Keunbong Do, Steven G Boxer
Split GFPs have been widely applied for monitoring protein-protein interactions by expressing GFPs as two or more constituent parts linked to separate proteins that only fluoresce on complementing with one another. Although this complementation is typically irreversible, it has been shown previously that light accelerates dissociation of a noncovalently attached β-strand from a circularly permuted split GFP, allowing the interaction to be reversible. Reversible complementation is desirable, but photodissociation has too low of an efficiency (quantum yield <1%) to be useful as an optogenetic tool...
March 14, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28242680/agrobacterium-delivered-virulence-protein-vire2-is-trafficked-inside-host-cells-via-a-myosin-xi-k-powered-er-actin-network
#11
Qinghua Yang, Xiaoyang Li, Haitao Tu, Shen Q Pan
Agrobacterium tumefaciens causes crown gall tumors on various plants by delivering transferred DNA (T-DNA) and virulence proteins into host plant cells. Under laboratory conditions, the bacterium is widely used as a vector to genetically modify a wide range of organisms, including plants, yeasts, fungi, and algae. Various studies suggest that T-DNA is protected inside host cells by VirE2, one of the virulence proteins. However, it is not clear how Agrobacterium-delivered factors are trafficked through the cytoplasm...
March 14, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28218346/usefulness-of-clearance-parametric-images-in-detection-of-regional-renal-parenchyma-dysfunction
#12
Jacek Kuśmierek, Małgorzata Bieńkiewicz, Tomasz Konecki, Marian Surma, Marek Sosnowski, Anna Płachcińska
BACKGROUND: The aim of the study was to examine whether parametric clearance images (PAR) enhance diagnostic potential of a dynamic renal scintigraphy with detection of local dysfunction of kidneys, on a model of kidneys after treatment with extracorporeal shock wave lithotripsy (ESWL), MATERIAL AND METHODS: Kidneys after ESWL were accepted as a proper model for the implementation of this objective because of the previously proven damaging effect of a shock wave on renal parenchyma and known region of ESWL application...
2017: Nuclear Medicine Review. Central & Eastern Europe
https://www.readbyqxmd.com/read/28212417/splitax-a-novel-method-to-assess-the-function-of-engineered-nucleases
#13
Richard A Axton, Sharmin S Haideri, Martha Lopez-Yrigoyen, Helen A Taylor, Lesley M Forrester
Engineered nucleases have been used to generate knockout or reporter cell lines and a range of animal models for human disease. These new technologies also hold great promise for therapeutic genome editing. Current methods to evaluate the activity of these nucleases are time consuming, require extensive optimization and are hampered by readouts with low signals and high background. We have developed a simple and easy to perform method (SplitAx) that largely addresses these issues and provides a readout of nuclease activity...
2017: PloS One
https://www.readbyqxmd.com/read/28108276/hydrogen-sulfide-reduces-rage-toxicity-through-inhibition-of-its-dimer-formation
#14
Hong Zhou, Lei Ding, Zhiyuan Wu, Xu Cao, Qichun Zhang, Li Lin, Jin-Song Bian
RAGE is important in the development of neurodegenerative diseases. The present study was designed to investigate the effect of hydrogen sulfide (H2S, an endogenous gaseous mediator) on the cytotoxicity caused by RAGE activation during the chronic oxidative stress. Aβ1-42 decreased cell viability and induced cell senescence in SH-SY5Y cells. Treatment with advanced glycation end products (AGEs) induced cell injury in HEK293 cells stably expressing RAGE (HEK293-RAGE) and stimulated inflammatory responses in SH-SY5Y cells...
March 2017: Free Radical Biology & Medicine
https://www.readbyqxmd.com/read/27943182/monitoring-synapses-via-trans-synaptic-gfp-complementation
#15
Theodoros Tsetsenis
Over the last years, the analysis of synaptic connectivity in the mammalian brain has been accelerated by the use of techniques combining electrophysiology, light microscopy, viral tracing, and genetic manipulations in animal models. Of particular interest are methods that aim to label synapses by tethering complementary split GFP fragments in opposing sites of the synaptic cleft. Here, I describe SynView, a method for monitoring synapse formation based on GFP complementation, and provide a detailed protocol for use in neuronal cultures from mouse hippocampus...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27915978/cell-compatibility-of-an-eposimal-vector-mediated-by-the-characteristic-motifs-of-matrix-attachment-regions
#16
Tian-Yun Wang, Li Wang, Yu-Xin Yang, Chun-Peng Zhao, Yan-Long Jia, Qin Li, Jun-He Zhang, Yi-You Peng, Miao Wang, Hong-Yan Xu, Xiao-Yin Wang
The characteristic sequence of β-interferon matrix attachment regions (MARs) can mediate transgene expression via episomal vectors in Chinese hamster ovary (CHO) cells. However, the host cells were from hamster ovaries, which are not suitable target cells for gene therapy. In this study, we aimed to evaluate the suitability of 12 different human cell lines as target cells for gene therapy. We transfected the cells with episomal vectors and obtained colonies stably expressing the vector products after G418 screening...
2016: Current Gene Therapy
https://www.readbyqxmd.com/read/27852847/visualizing-the-essential-role-of-complete-virion-assembly-machinery-in-efficient-hepatitis-c-virus-cell-to-cell-transmission-by-a-viral-infection-activated-split-intein-mediated-reporter-system
#17
Fanfan Zhao, Ting Zhao, Libin Deng, Dawei Lv, Xiaolong Zhang, Xiaoyu Pan, Jun Xu, Gang Long
Hepatitis C virus (HCV) infects 2 to 3% of the world population and is a leading cause of liver diseases such as fibrosis, cirrhosis, and hepatocellular carcinoma. Many aspects of HCV study, ranging from molecular virology and antiviral drug development to drug resistance profiling, were supported by straightforward assays of HCV replication and infection. Among these assays, the HCV-dependent fluorescence relocalization (HDFR) system allowed live-cell visualization of infection without modifying the viral genome, but this strategy required careful recognition of the fluorescence relocalization pattern for its high fluorescence background in the cytoplasm...
January 15, 2017: Journal of Virology
https://www.readbyqxmd.com/read/27831485/analysis-of-membrane-proteins-localizing-to-the-inner-nuclear-envelope-in-living-cells
#18
Christine J Smoyer, Santharam S Katta, Jennifer M Gardner, Lynn Stoltz, Scott McCroskey, William D Bradford, Melainia McClain, Sarah E Smith, Brian D Slaughter, Jay R Unruh, Sue L Jaspersen
Understanding the protein composition of the inner nuclear membrane (INM) is fundamental to elucidating its role in normal nuclear function and in disease; however, few tools exist to examine the INM in living cells, and the INM-specific proteome remains poorly characterized. Here, we adapted split green fluorescent protein (split-GFP) to systematically localize known and predicted integral membrane proteins in Saccharomyces cerevisiae to the INM as opposed to the outer nuclear membrane. Our data suggest that components of the endoplasmic reticulum (ER) as well as other organelles are able to access the INM, particularly if they contain a small extraluminal domain...
November 21, 2016: Journal of Cell Biology
https://www.readbyqxmd.com/read/27742884/high-silicon-accumulation-in-the-shoot-is-required-for-down-regulating-the-expression-of-si-transporter-genes-in-rice
#19
Namiki Mitani-Ueno, Naoki Yamaji, Jian Feng Ma
Rice requires high silicon (Si) for its high and sustainable yield. The efficient uptake of Si in rice is mediated by two transporters OsLsi1 and OsLsi2, which function as influx and efflux transporters, respectively. Our previous studies showed that the mRNA expression levels of these transporter genes were down-regulated by Si. Herein we investigated the mechanism underlying regulation of OsLsi1 and OsLsi2 expression. There was a negative correlation between the expression level of OsLsi1 and OsLsi2 and shoot Si accumulation when the rice seedlings were exposed to different Si supply conditions...
December 2016: Plant & Cell Physiology
https://www.readbyqxmd.com/read/27608276/split-gfp-sers-enhancers-in-plasmonic-nanocluster-probes
#20
Taerin Chung, Tugba Koker, Fabien Pinaud
The assembly of plasmonic metal nanoparticles into hot spot surface-enhanced Raman scattering (SERS) nanocluster probes is a powerful, yet challenging approach for ultrasensitive biosensing. Scaffolding strategies based on self-complementary peptides and proteins are of increasing interest for these assemblies, but the electronic and the photonic properties of such hybrid nanoclusters remain difficult to predict and optimize. Here, split-green fluorescence protein (sGFP) fragments are used as molecular glue and the GFP chromophore is used as a Raman reporter to assemble a variety of gold nanoparticle (AuNP) clusters and explore their plasmonic properties by numerical modeling...
September 8, 2016: Small
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