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Protein expression and purification

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https://www.readbyqxmd.com/read/29161226/development-of-competitive-enzyme-linked-immunosorbent-assays-for-antibody-detection-based-on-bluetongue-virus-monoclonal-antibodies
#1
Zhou Han, Zang Mingxin, Li Xuechun, Xu Yigang, Qiao Xinyuan, Wang Li, Cui Wen, Jiang Yanping, Li Yijing, Tang Lijie
Bluetongue is a ruminant infectious disease that is characterized by hyperpyrexia, leukocyte decrease and mucosal ulcerative inflammation changes. In this study, three segments of Bluetongue virus (BTV)-8 VP2 protein (BTV-8A, 8B, and 8C) were cloned into pET-28a (+) and pMAl-c5X vectors and expressed in Escherichia coli BL21 (DE3) as histidine (His)-tagged (His-8A/8B/8C) and maltose-binding protein (MBP)-tagged (MBP-8A/8B/8C) fusion proteins. After purification, His-8A/8B/8C were used to immunize BALB/mice and MBP-8A/8B/8C were used to screen for monoclonal antibody (mAb)-secreting hybridomas...
November 21, 2017: Viral Immunology
https://www.readbyqxmd.com/read/29160698/functional-characterization-of-salicylic-acid-carboxyl-methyltransferase-from-camellia-sinensis-providing-the-aroma-compound-of-methyl-salicylate-during-withering-process-of-white-tea
#2
Wei-Wei Deng, Rongxiu Wang, Tianyuan Yang, Li'na Jiang, Zhengzhu Zhang
Methyl salicylate (MeSA) is one of the volatile organic compounds (VOCs) that releases floral scent, and plays an important role in tea sweet flowery aroma. During the withering process for white tea producing, MeSA was generated by salicylic acid carboxyl methyltransferase (SAMT) with salicylic acid (SA), and the specific floral scent was formed. In this study, we firstly cloned a CsSAMT from tea leaves (GenBank accession No. MG459470) and used Escherichia coli and Saccharomyces cerevisiae to express the recombinant CsSAMT...
November 21, 2017: Journal of Agricultural and Food Chemistry
https://www.readbyqxmd.com/read/29159916/purification-and-characterization-of-ribosomal-proteins-l27-and-l30-having-antimicrobial-activity-produced-by-the-lactobacillus-salivarius-sgl03
#3
Paolo Pidutti, Federica Federici, Jessica Brandi, Laura Manna, Eleonora Rizzi, Umberto Marini, Daniela Cecconi
AIMS: The aim of this study was to investigate the antimicrobial potential of proteins secreted by a new strain of Lactobacillus salivarius. METHODS AND RESULTS: The secretome of L. salivarius SGL03 strain was analyzed by gel-assisted fractionation and MS/MS to identify low-molecular-mass proteins. This strategy allowed us to identify ten secreted proteins. Then a combination of heterologous expression and agar well diffusion was used to characterize them as to their antimicrobial activity, mechanisms of action, and stability...
November 21, 2017: Journal of Applied Microbiology
https://www.readbyqxmd.com/read/29155755/in-vivo-detection-and-analysis-of-rb-protein-sumoylation-in-human-cells
#4
Fengxi Meng, Xiaofeng Li, Hui Ren, Jiang Qian
The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions...
November 2, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29155144/bay-60-2770-activates-two-isoforms-of-nitric-oxide-sensitive-guanylyl-cyclase-evidence-for-stable-insertion-of-activator-drugs
#5
Anne Sömmer, Peter Sandner, Sönke Behrends
Nitric oxide sensitive guanylyl cyclase (NOsGC), a hemoprotein and the major physiological receptor for nitric oxide (NO), is a heterodimer with the α1/β1 and α2/β1 isoforms known to be important for NO-signaling and conversion of GTP to cGMP in humans. Two innovative classes of compounds modulating the NO/cGMP signaling pathway have been discovered: the heme-dependent sGC stimulators, that stimulate NOsGC directly and also increase the affinity towards NO, and the heme-independent sGC activators, that are thought to bind to oxidized and heme-free NOsGC in tissues exposed to oxidative stress...
November 15, 2017: Biochemical Pharmacology
https://www.readbyqxmd.com/read/29151220/high-efficiency-expression-of-yeast-derived-g-protein-coupled-receptors-and-19-f-labeling-for-dynamical-studies
#6
Libin Ye, Alexander P Orazietti, Aditya Pandey, R Scott Prosser
We describe a detailed protocol for heterologous expression of the human adenosine A2A G-protein coupled receptor (GPCR), using Pichia pastoris. Details are also provided for the reconstitution and functional purification steps. Yields of 2-6 mg/g membrane were obtained prior to functional purification (ligand column purification). Typically, functional purification reduced overall yields by a factor of 2-4, resulting in final functional production of 0.5-3 mg/L membrane. Yeast is an excellent protein expression system for NMR given its high tolerance for isotope-enriched solvents and its ability to grow in minimal media...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29149798/reconstructing-biosynthetic-pathway-of-the-plant-derived-cancer-chemopreventive-precursor-glucoraphanin-in-escherichia-coli
#7
Han Yang, Feixia Liu, Yin Li, Bo Yu
Epidemiological data confirmed a strong correlation between regular consumption of cruciferous vegetables and lower cancer risk. This cancer preventive property is mainly attributed to the glucosinolate products, such as glucoraphanin found in broccoli that is derived from methionine. Here we report the first successful reconstruction of the complete biosynthetic pathway of glucoraphanin from methionine in Escherichia coli via gene selection, pathway design, and protein engineering. We used branched-chain amino transferase 3 to catalyze two transamination steps to ensure the purity of precursor molecules and used cysteine as a sulfur donor to simplify the synthesis pathway...
November 17, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/29146152/improved-antifungal-activity-of-barley-derived-chitinase-i-gene-that-overexpress-a-32kda-recombinant-chitinase-in-escherichia-coli-host
#8
Nida Toufiq, Bushra Tabassum, Muhammad Umar Bhatti, Anwar Khan, Muhammad Tariq, Naila Shahid, Idrees Ahmad Nasir, Tayyab Husnain
Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L.) cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935bp full-length chitinase I gene...
October 31, 2017: Brazilian Journal of Microbiology: [publication of the Brazilian Society for Microbiology]
https://www.readbyqxmd.com/read/29143228/effect-of-exosome-biomarkers-for-diagnosis-and-prognosis-of-breast-cancer-patients
#9
REVIEW
M Wang, S Ji, G Shao, J Zhang, K Zhao, Z Wang, A Wu
PURPOSE: Exosomes are gradually detected as an indicator for diagnosis and prognosis of breast cancer in clinic and a systematic review was conducted. METHODS: A search for clinical studies published before July 1, 2017 was performed. Methods of exosome purification and identification from all studies were extracted. For diagnosis evaluation, the comparison of exosome biomarkers expression between breast cancer patients and healthy women was obtained; for prognosis prediction, the correlation between exosome biomarkers expression and chemotherapy resistance, overall survival (OS), disease-free survival (DFS), recurrence and metastasis of breast cancer was also extracted...
November 15, 2017: Clinical & Translational Oncology
https://www.readbyqxmd.com/read/29143175/vectors-for-expression-of-signal-peptide-dependent-proteins-in-baculovirus-insect-cell-systems-and-their-application-to-expression-and-purification-of-the-high-affinity-immunoglobulin-gamma-fc-receptor-i-in-complex-with-its-gamma-chain
#10
Le T M Le, Jens R Nyengaard, Monika M Golas, Bjoern Sander
Integral membrane proteins play a central role in various cellular functions and are important therapeutic targets. However, technical challenges in the overexpression and purification of membrane proteins often represent a limiting factor for biochemical and structural studies. Here, we constructed a set of vectors, derivatives of MultiBac vectors that can be used to express proteins with a cleavable N-terminal signal peptide in insect cells. We propose these vectors for expression of type I membrane proteins and other secretory pathway proteins that require the signal recognition particle for translocation to the endoplasmic reticulum (ER)...
November 15, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/29139018/vaccination-against-hydatidosis-molecular-cloning-and-optimal-expression-of-the-eg95nc-recombinant-antigen-in-escherichia-coli
#11
M Jazouli, M Lightowlers, C G Gauci, K Tadlaoui, A Belmlih, M M Ennaji, M Elharrak
Cystic echinococcosis (CE) is a widely distributed zoonosis that is highly endemic in the Mediterranean basin. The disease represents a serious public health threat and causes economic losses. The parasite life-cycle involves dogs and ruminants as definitive and intermediate hosts; humans are accidently infected, causing serious clinical issues. Vaccination of ruminants and dog treatments represent the most efficient measures to prevent parasite transmission. The recombinant protein vaccine, EG95, has been used successfully in sheep vaccine trials against CE in several countries...
November 14, 2017: Protein Journal
https://www.readbyqxmd.com/read/29133437/abnormal-microglia-and-enhanced-inflammation-related-gene-transcription-in-mice-with-conditional-deletion-of-ctcf-in-camk2a-cre-expressing-neurons
#12
Bryan E McGill, Ruteja A Barve, Susan E Maloney, Amy Strickland, Nicholas Rensing, Peter Wang, Michael Wong, Richard Head, David F Wozniak, Jeffrey Milbrandt
CCCTC-binding factor (CTCF) is an 11 zinc finger DNA-binding domain protein that regulates gene expression by modifying three dimensional chromatin structure. Human mutations in CTCF cause intellectual disability and autistic features. Knocking out Ctcf in mouse embryonic neurons is lethal by neonatal age, but the effects of CTCF deficiency in postnatal neurons are less well studied. We knocked out Ctcf postnatally in glutamatergic forebrain neurons under the control of Camk2a-CreCtcf(loxP/loxP) ;Camk2a-Cre+ (Ctcf CKO) mice of both sexes were viable and exhibited profound deficits in spatial learning/memory, impaired motor co-ordination, and decreased sociability by 4 months of age...
November 13, 2017: Journal of Neuroscience: the Official Journal of the Society for Neuroscience
https://www.readbyqxmd.com/read/29132366/prokaryotic-expression-and-characterization-of-the-heterodimeric-construction-of-znt8-and-its-application-for-autoantibodies-detection-in-diabetes-mellitus
#13
Natalia I Faccinetti, Luciano L Guerra, Adriana V Sabljic, Silvina S Bombicino, Bruno D Rovitto, Ruben F Iacono, Edgardo Poskus, Aldana Trabucchi, Silvina N Valdez
BACKGROUND: In the present work we described the recombinant production and characterization of heterodimeric construction ZnT8-Arg-Trp325 fused to thioredoxin using a high-performance expression system such as Escherichia coli. In addition, we apply this novel recombinant antigen in a non-radiometric method, with high sensitivity, low operational complexity and lower costs. RESULTS: ZnT8 was expressed in E. coli as a fusion protein with thioredoxin (TrxZnT8). After 3 h for induction, recombinant protein was obtained from the intracellular soluble fraction and from inclusion bodies and purified by affinity chromatography...
November 13, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/29131146/synthetic-microbial-consortia-enable-rapid-assembly-of-pure-translation-machinery
#14
Fernando Villarreal, Luis E Contreras-Llano, Michael Chavez, Yunfeng Ding, Jinzhen Fan, Tingrui Pan, Cheemeng Tan
Assembly of recombinant multiprotein systems requires multiple culturing and purification steps that scale linearly with the number of constituent proteins. This problem is particularly pronounced in the preparation of the 34 proteins involved in transcription and translation systems, which are fundamental biochemistry tools for reconstitution of cellular pathways ex vivo. Here, we engineer synthetic microbial consortia consisting of between 15 and 34 Escherichia coli strains to assemble the 34 proteins in a single culturing, lysis, and purification procedure...
November 13, 2017: Nature Chemical Biology
https://www.readbyqxmd.com/read/29130213/systematic-detection-of-poly-a-rna-interacting-proteins-and-their-differential-binding
#15
Miha Milek, Markus Landthaler
RNA-binding proteins are dynamic posttranscriptional regulators of gene expression. Identification of mRNA-binding proteins in a given experimental setting is thus of great importance. We describe a procedure to enrich for direct poly(A)(+) RNA protein binders by 4-thiouridine-enhanced UV cross-linking and oligo(dT) purification. Subsequent nuclease-mediated release of RNA-binding proteins (RBPs) from mRNA allows for detection of eluted proteins by mass spectrometry. In addition, we provide a comparative approach to detect differences in RBP binding activity upon a biological stimulus...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29130191/axon-trap-ribotag-affinity-purification-of-translated-mrnas-from-neuronal-axons-in-mouse-in-vivo
#16
Toshiaki Shigeoka, Jane Jung, Christine E Holt, Hosung Jung
Translating ribosome affinity purification (TRAP) is a widely used technique to analyze ribosome-bound mRNAs in particular target cells that express a tagged ribosomal protein. We developed axon-TRAP-RiboTag, a TRAP-based method that allows purification and identification of translated mRNAs from distal neuronal axons in mouse, and identified more than 2000 of translated mRNAs in retinal ganglion cell (RGC) axons in vivo. The use of Cre-negative littermate control to filter out false-positive signals allows unbiased detection, and combining TRAP with in vitro ribosome run-off enables identification of actively translated mRNAs...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29129402/hepatitis-b-core-antigen-based-novel-vaccine-against-porcine-epidemic-diarrhea-virus
#17
Frank Gillam, Jianqiang Zhang, Chenming Zhang
Porcine epidemic diarrhea Virus (PEDV) is the causative agent of porcine epidemic diarrhea, which is a devastating viral disease and causes severe economic loss to the swine industry. Current vaccine options for PEDV include modified live viruses and killed live viruses. Though these vaccines have shown efficacy, some have side effects including viral shedding. This report details an E. coli based expression and purification process of multiple vaccine candidates for PEDV using Hepatitis B Core Antigen (HBcAg) as a backbone protein...
November 9, 2017: Journal of Virological Methods
https://www.readbyqxmd.com/read/29126399/production-and-purification-of-chimeric-hbc-virus-like-particles-carrying-influenza-virus-lah-domain-as-vaccine-candidates
#18
Andris Kazaks, I-Na Lu, Sophie Farinelle, Alex Ramirez, Vincenzo Crescente, Benjamin Blaha, Olotu Ogonah, Tarit Mukhopadhyay, Mapi Perez de Obanos, Alejandro Krimer, Inara Akopjana, Janis Bogans, Velta Ose, Anna Kirsteina, Tatjana Kazaka, Nicola J Stonehouse, David J Rowlands, Claude P Muller, Kaspars Tars, William M Rosenberg
BACKGROUND: The lack of a universal influenza vaccine is a global health problem. Interest is now focused on structurally conserved protein domains capable of eliciting protection against a broad range of influenza virus strains. The long alpha helix (LAH) is an attractive vaccine component since it is one of the most conserved influenza hemagglutinin (HA) stalk regions. For an improved immune response, the LAH domain from H3N2 strain has been incorporated into virus-like particles (VLPs) derived from hepatitis B virus core protein (HBc) using recently developed tandem core technology...
November 10, 2017: BMC Biotechnology
https://www.readbyqxmd.com/read/29121909/successful-production-of-the-potato-antimicrobial-peptide-snakin-1-in-baculovirus-infected-insect-cells-and-development-of-specific-antibodies
#19
Natalia Inés Almasia, María Paula Molinari, Guillermo Andrés Maroniche, Vanesa Nahirñak, María Pilar Barrios Barón, Oscar Alberto Taboga, Cecilia Vazquez Rovere
BACKGROUND: Snakin-1 (StSN1) is a broad-spectrum antimicrobial cysteine-rich peptide isolated from Solanum tuberosum. Its biotechnological potential has been already recognized since it exhibits in vivo antifungal and antibacterial activity. Most attempts to produce StSN1, or homologous peptides, in a soluble native state using bacterial, yeast or synthetic expression systems have presented production bottlenecks such as insolubility, misfolding or low yields. RESULTS: In this work, we successfully expressed a recombinant StSN1 (rSN1) in Spodoptera frugiperda (Sf9) insect cells by optimizing several of the parameters for its expression in the baculovirus expression system...
November 9, 2017: BMC Biotechnology
https://www.readbyqxmd.com/read/29121065/proteomic-analysis-reveals-novel-ligands-and-substrates-for-lnx1-e3-ubiquitin-ligase
#20
Joan A Lenihan, Orthis Saha, Paul W Young
Ligand of Numb protein X1 (LNX1) is an E3 ubiquitin ligase that contains a catalytic RING (Really Interesting New Gene) domain and four PDZ (PSD-95, DlgA, ZO-1) domains. LNX1 can ubiquitinate Numb, as well as a number of other ligands. However, the physiological relevance of these interactions in vivo remain unclear. To gain functional insights into the LNX family, we have characterised the LNX1 interactome using affinity purification and mass spectrometry. This approach identified a large number of novel LNX1-interacting proteins, as well as confirming known interactions with NUMB and ERC2...
2017: PloS One
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