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GFP split

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
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 of 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...
April 7, 2017: Journal of Biological Chemistry
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
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
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
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
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
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
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
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...
December 2, 2016: Current Gene Therapy
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
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 9, 2016: Journal of Cell Biology
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
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
Peter Lönn, Apollo D Kacsinta, Xian-Shu Cui, Alexander S Hamil, Manuel Kaulich, Khirud Gogoi, Steven F Dowdy
Bioactive macromolecular peptides and oligonucleotides have significant therapeutic potential. However, due to their size, they have no ability to enter the cytoplasm of cells. Peptide/Protein transduction domains (PTDs), also called cell-penetrating peptides (CPPs), can promote uptake of macromolecules via endocytosis. However, overcoming the rate-limiting step of endosomal escape into the cytoplasm remains a major challenge. Hydrophobic amino acid R groups are known to play a vital role in viral escape from endosomes...
September 8, 2016: Scientific Reports
Florian S Frueh, Thomas Später, Nicole Lindenblatt, Maurizio Calcagni, Pietro Giovanoli, Claudia Scheuer, Michael D Menger, Matthias W Laschke
Full-thickness skin defects can be covered with dermal skin substitutes in combination with split-thickness skin grafts. However, slow vascularization of the matrices bears the risk of wound infection and extends the length of hospitalization. To overcome these problems, we describe a promising vascularization strategy. Green fluorescent protein(+) adipose tissue-derived microvascular fragments (ad-MVF) were isolated from epididymal fat pads of C57BL/6-Tg(CAG-EGFP)1Osb/J mice. ad-MVF were seeded on collagen-glycosaminoglycan matrices, which were implanted into full-thickness skin defects in the dorsal skinfold chamber of wild-type C57BL/6 mice...
January 2017: Journal of Investigative Dermatology
Christoph Priesner, Krasimira Aleksandrova, Ruth Esser, Nadine Mockel-Tenbrinck, Jana Leise, Katharina Drechsel, Michael Marburger, Andrea Quaiser, Lilia Goudeva, Lubomir Arseniev, Andrew Kaiser, Wolfgang Glienke, Ulrike Koehl
Multiple clinical studies demonstrated that adaptive immunotherapy using redirected T cells against advanced cancer has led to promising results with improved survival of the patients. The continuously increasing interest in those advanced Gene Therapy Medicinal Products (GTMPs) leads to a challenge on the manufacturing side regarding automation, process robustness and cell storage. Therefore, our study addresses the proof of principle in clinical-scale selection, stimulation, transduction and expansion of T cells using the automated closed CliniMACS® Prodigy system...
August 25, 2016: Human Gene Therapy
Dorthe Rippert, Jürgen J Heinisch
Septins are key components of the cell division machinery from yeast to humans. The model yeast Saccharomyces cerevisiae has five mitotic septins, Cdc3, Cdc10, Cdc11, Cdc12, and Shs1. Here we characterized the five orthologs from the genetically less-redundant milk yeast Kluyveromyces lactis. We found that except for KlSHS1 all septin genes are essential. Klshs1 deletions displayed temperature-sensitive growth and morphological defects. Heterologous complementation analyses revealed that all five K. lactis genes encode functional orthologs of their S...
September 2016: Fungal Genetics and Biology: FG & B
H M van der Schaar, C E Melia, J A C van Bruggen, J R P M Strating, M E D van Geenen, A J Koster, M Bárcena, F J M van Kuppeveld
Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recombinant enteroviruses that carry genes that encode RO-anchored viral proteins tagged with fluorescent reporters have not been reported thus far. To overcome this limitation, we used a split green fluorescent protein (split-GFP) system, comprising a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 residues...
July 2016: MSphere
Gregory C Finnigan, Angela Duvalyan, Elizabeth N Liao, Aspram Sargsyan, Jeremy Thorner
Various methods can provide a readout of the physical interaction between two biomolecules. A recently described tripartite split-GFP system has the potential to report by direct visualization via a fluorescence signal the intimate association of minimally tagged proteins expressed at their endogenous level in their native cellular milieu and can capture transient or weak interactions. Here we document the utility of this tripartite split-GFP system to assess in living cells protein-protein interactions in a dynamic cytoskeletal structure-the septin collar at the yeast bud neck...
September 1, 2016: Molecular Biology of the Cell
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