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https://www.readbyqxmd.com/read/28736549/cloning-expression-isotope-labeling-and-purification-of-transmembrane-protein-merf-from-mercury-resistant-enterobacter-sp-az-15-for-nmr-studies
#1
Aatif Amin, Zakia Latif
Mercury resistant (Hg(R)) Enterobacter sp. AZ-15 was isolated from heavy metal polluted industrial wastewater samples near to districts Kasur and Sheikhupura, Pakistan. 16S rDNA ribotyping and phylogentic analysis showed 98% homology with already reported Enterobacter species. The merF gene encoding transmembrane protein-MerF was amplified from genomic DNA and ligated into pET31b+ vector using restriction endonucleases, SphI and XhoI. The genetic codons of merF gene encoding cysteine residues were mutated into codons, translating into serine residues by site-directed mutagenesis...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28736314/expression-and-purification-of-single-cysteine-containing-mutant-variants-of-the-mouse-prion-protein-by-oxidative-refolding
#2
Ishita Sengupta, Jayant B Udgaonkar
The folding and aggregation of proteins has been studied extensively, using multiple probes. To facilitate such experiments, introduction of spectroscopically-active moieties in to the protein of interest is often necessary. This is commonly achieved by specifically labelling cysteine residues in the protein, which are either present naturally or introduced artificially by site-directed mutagenesis. In the case of the recombinant prion protein, which is normally expressed in inclusion bodies, the presence of the native disulfide bond complicates the correct refolding of single cysteine-containing mutant variants of the protein...
July 20, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28735865/rps27a-enhances-ebv-encoded-lmp1-mediated-proliferation-and-invasion-by-stabilizing-of-lmp1
#3
Seung-Woo Hong, Seung-Mi Kim, Dong-Hoon Jin, Yeong Seok Kim, Dae Young Hur
Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is an oncoviral protein that plays a pivotal role in EBV-induced oncogenic transformation. The function of LMP1 in EBV-induced oncogenesis has been well studied. However, the molecular mechanisms underlying LMP1 protein stability remain poorly understood. In this study, we found that ribosomal protein s27a (RPS27a) regulates LMP1 stability by a tandem affinity purification analysis. RPS27a interacts directly with LMP1 in vitro and in vivo. Furthermore, overexpression of RPS27a increases the half-life of LMP1 in 293T cells, whereas downregulation of RPS27a using lentiviral shRNA technology accelerates the decrease in LMP1 protein level in EBV-transformed B cells...
July 20, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/28735093/stable-isotope-labeling-by-fatty-acids-in-cell-culture-silfac-coupled-with-isotope-pattern-dependent-mass-spectrometry-for-global-screening-of-lipid-hydroperoxide-mediated-protein-modifications
#4
Ryo Takahashi, Shuhei Fujioka, Tomoyuki Oe, Seon Hwa Lee
Lipid hydroperoxide-mediated modifications of proteins are receiving increasing attention because of their possible involvement in various degenerative diseases. These biological effects are attributed to the ability of lipid peroxidation-derived aldehydes to react with the nucleophilic sites of proteins. Here we describe a methodology involving metabolic labeling coupled with mass spectrometry-based proteomic analysis that enables global screening of lipid hydroperoxide-mediated protein modifications in a cell system...
July 19, 2017: Journal of Proteomics
https://www.readbyqxmd.com/read/28735080/recombinant-truncated-e-protein-as-a-new-vaccine-candidate-against-nontypeable-h-influenzae-its-expression-and-immunogenic-evaluation
#5
Ava Behrouzi, Saeid Bouzari, Farzam Vaziri, Abolfazl Fateh, Parviz Afrough, Atefeh Davoudi Vijeh Motlagh, Seyed Davar Siadat
Protein E (PE) is a conserved entity observed in both nontypeable Haemophilus influenzae (NTHi) and encapsulated H. influenzae. This is a small surface lipoprotein, consisting of only 160 amino acids, involved in the adhesion of H. influenzae to various types of epithelial cells. A 384-bp-long fragment from NTHi PE was cloned into the prokaryotic expression vector pBAD-gIIIA. The recombinant protein was expressed with arabinose and then purified by affinity purification on an Ni-NTA agarose matrix. BALB/c mice were immunized by subcutaneous injection with purified recombinant truncated PE mixed with an alum adjuvant...
July 19, 2017: Microbial Pathogenesis
https://www.readbyqxmd.com/read/28734840/an-improved-purification-method-for-the-lysosomal-storage-disease-protein-%C3%AE-glucuronidase-produced-in-cho-cells
#6
Erica J Fratz-Berilla, Stephanie A Ketcham, Hamideh Parhiz, Muhammad Ashraf, Chikkathur N Madhavarao
Human β-glucuronidase (GUS; EC 3.2.1.31) is a lysosomal enzyme that catalyzes the hydrolysis of β-d-glucuronic acid residues from the non-reducing termini of glycosaminoglycans. Impairment in GUS function leads to the metabolic disorder mucopolysaccharidosis type VII, also known as Sly syndrome. We produced GUS from a CHO cell line grown in suspension in a 15 L perfused bioreactor and developed a three step purification procedure that yields ∼99% pure enzyme with a recovery of more than 40%. The method can be completed in two days and has the potential to be integrated into a continuous manufacturing scheme...
July 19, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28734838/expression-purification-and-characterization-of-sll1981-protein-from-cyanobacterium-synechocystis-sp-pcc6803
#7
Xiaoqin Wang, Guofeng Lei, Xiaoyu Wu, Fei Wang, Chongde Lai, Zhimin Li
The sll1981 protein from cyanobacterium Synechocystis sp. PCC6803 had been reported to exhibit acetolactate synthase (ALS) and L-myo-inositol-1-phosphate synthase (MIPS) activities previously. Based on amino acids sequences alignment, sll1981 protein was postulated to function as α-ketoglutarate decarboxylase (α-KGD), which played important role in completing cyanobacterial tricarboxylic acid (TCA) cycle. However the detailed enzymatic kinetics of sll1981 as ALS, MIPS and α-KGD were not determined yet. In this study, the recombinant sll1981 protein was purified from supernatant of E...
July 19, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28734361/preparation-of-intact-mitochondria-using-free-flow-isoelectric-focusing-with-post-ph-gradient-sample-injection-for-morphological-functional-and-proteomics-studies
#8
Yu-Chen He, Fan-Zhi Kong, Liu-Yin Fan, Jane Y Wu, Xiao-Ping Liu, Jun Li, Yan Sun, Qiang Zhang, Ying Yang, Xue-Jing Wu, Hua Xiao, Cheng-Xi Cao
Mitochondria play essential roles in both energy metabolism and cell signaling, which are critical for cell survival. Although significant efforts have been invested in understanding mitochondrial biology, methods for intact mitochondria preparation are technically challenging and remain to be improved. New methods for heterogeneous mitochondria purification will therefore boost our understanding on their physiological and biophysical properties. Herein, we developed a novel recycling free-flow isoelectric focusing (RFFIEF) with post-pH gradient sample injection (post-PGSI) for preparative separation of mitochondria...
August 22, 2017: Analytica Chimica Acta
https://www.readbyqxmd.com/read/28734049/development-of-aptamers-against-unpurified-proteins
#9
Shinichi Goto, Kaori Tsukakoshi, Kazunori Ikebukuro
SELEX (Systematic Evolution of Ligands by EXponential enrichment) has been widely used for the generation of aptamers against target proteins. However, its requirement for pure target proteins remains a major problem in aptamer selection, as procedures for protein purification from crude bio-samples are not only complicated but also time and labour consuming. This is because native proteins can be found in a large number of diverse forms because of posttranslational modifications and their complicated molecular conformations...
July 22, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28733289/an-enterotoxin-like-binary-protein-from-pseudomonas-protegens-with-potent-nematicidal-activity
#10
Jun-Zhi Wei, Daniel L Siehl, Zhenglin Hou, Barbara Rosen, Jarred Oral, Christopher G Taylor, Gusui Wu
Soil microbes are a major food source for free-living soil nematodes. It is known that certain soil bacteria have evolved systems to combat predation. We identified the nematode-antagonistic Pseudomonas protegens strain 15G2 from screening of microbes. Through protein purification we identified a binary protein, designated as Pp-ANP, that is responsible for the nematicidal activity. This binary protein inhibits Caenorhabditis elegans growth and development by arresting larvae at L1 stage and killing older staged worms...
July 21, 2017: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/28732905/preparation-of-fungus-derived-chitin-nanocrystals-and-their-dispersion-stability-evaluation-in-aqueous-media
#11
Ning Lin, Shasha Zhao, Lin Gan, Peter R Chang, Tao Xia, Jin Huang
The chitin nanocrystal is a promising nano-reinforcing agent, but the parasitic pathogens carried on crabs and shrimp shells as main sources limit its application in some fields. In this study, the ChNs which avoided possible safety risks were extracted from mushrooms via protein/mineral-purification and subsequent HCl-hydrolysis. Such fungus-derived ChNs presented an α-chitin crystalline structure with a length of 143±24nm and a diameter of 10±2nm. Since the dispersion stability of ChNs suspension determines their further applications, this present study emphasized the dispersity of ChNs in aqueous media evaluated by the viscosity under steady-shear flow and UV-vis absorption, whose results indicated that ChNs in dispersion would aggregate when the concentration of homogeneous dispersion reached 0...
October 1, 2017: Carbohydrate Polymers
https://www.readbyqxmd.com/read/28732888/separation-of-polysaccharides-from-spirulina-platensis-by-hsccc-with-ethanol-ammonium-sulfate-atps-and-their-antioxidant-activities
#12
Xueyan Wu, Ruichang Li, Yongjie Zhao, Yang Liu
Three separation methods for water-soluble polysaccharides from Spirulina platensis (PSP) were compared, including sevage deproteinization and column chromatography, ethanol-ammonium sulfate aqueous two-phase system (ATPS) and column chromatography, and one-step high speed counter-current chromatography (HSCCC) with ethanol-ammonium sulfate ATPS. ATPS was confirmed as an efficient alternative method of protein removal for PSP purification. For the HSCCC with ethanol-ammonium sulfate ATPS, the stationary retention reached to 50...
October 1, 2017: Carbohydrate Polymers
https://www.readbyqxmd.com/read/28732682/characterization-and-purification-of-proteins-suitable-for-the-production-of-antibodies-against-ca-liberibacter-asiaticus
#13
Huawei Liu, Sagheer Atta, John S Hartung
The citrus disease huanglongbing (HLB), which is caused by 'Candidatus Liberibacter asiaticus' (CaLas), is one of the most devastating pathogens of citrus, and with no effective method of control, poses a serious threat to citrus production throughout the world. In a previous study we described the production of single chain antibodies against several CaLas proteins that provide the basis for efficient and accurate detection of CaLas in citrus tissues. The isolation of a sufficient amount of purified antigen is a key step in the production of functional antibodies...
July 18, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28731574/rapid-buffer-and-ligand-screening-for-affinity-chromatography-by-multiplexed-surface-plasmon-resonance-imaging
#14
Karin P M Geuijen, Daniëlle E J W van Wijk-Basten, David F Egging, Richard B M Schasfoort, Michel H Eppink
Protein purifications are often based on the principle of affinity chromatography, where the protein of interest selectively binds to an immobilized ligand. The development of affinity purification requires selecting proper wash and elution conditions. In recent years, miniaturization of the purification process is applied to speed up the development (e.g. microtiterplates, robocolumns). We have studied the application of surface plasmon resonance imaging (SPRi) as a tool to simultaneously screen many buffer conditions for wash and elution steps in an affinity-based purification process...
July 21, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28731571/model-based-investigation-on-the-mass-transfer-and-adsorption-mechanisms-of-mono-pegylated-lysozyme-in-ion-exchange-chromatography
#15
Josefine Morgenstern, Gang Wang, Pascal Baumann, Jürgen Hubbuch
Recent studies highlighted the potential of PEGylated proteins to improve stabilities and pharmacokinetics of protein drugs. Ion-exchange chromatography (IEX) is among the most frequently used purification methods for PEGylated proteins. However, the underlying physical mechanisms allowing for a separation of different PEGamers (proteins with a varying number of attached PEG molecules) are not yet fully understood. In this work, mechanistic chromatography modeling was applied to gain a deeper understanding of the mass transfer and adsorption/desorption mechanisms of mono-PEGylated proteins in IEX...
July 21, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28731187/isolation-and-purification-of-novel-peptides-derived-from-sepia-ink-effects-on-apoptosis-of-prostate-cancer-cell-pc%C3%A2-3
#16
Fangfang Huang, Yinwen Jing, Guofang Ding, Zuisu Yang
Novel prostate cancer therapeutics are in high demand. In order to identify potential therapeutic targets, protein from sepia ink was hydrolyzed by utilizing pepsin in an orthogonal array design. Pepsin hydrolysate (SH) obtained at optimal conditions exhibited the highest antitumor activity. Subsequently, a novel antitumor peptide, which was termed SHP, was isolated through ultrafiltration, gel filtration chromatography and reversed phase high‑performance liquid chromatography. The amino acid sequence of SHP was identified as Leu‑Lys‑Glu‑Glu‑Asn‑Arg‑Arg‑Arg‑Arg‑Asp with a molecular mass of 1371...
July 21, 2017: Molecular Medicine Reports
https://www.readbyqxmd.com/read/28730304/novel-ribonuclease-activity-of-cusativin-from-cucumis-sativus-for-mapping-nucleoside-modifications-in-rna
#17
Balasubrahmanyam Addepalli, Sarah Venus, Priti Thakur, Patrick A Limbach
A recombinant ribonuclease, cusativin, was characterized for its cytidine-specific cleavage ability of RNA to map chemical modifications. Following purification of native cusativin protein as described before (Rojo et al. Planta 194:328, 17), partial amino acid sequencing was carried out to identify the corresponding protein coding gene in cucumber genome. Cloning and heterologous expression of the identified gene in Escherichia coli resulted in successful production of active protein as a C-terminal His-tag fusion protein...
July 20, 2017: Analytical and Bioanalytical Chemistry
https://www.readbyqxmd.com/read/28730213/magnetic-separation-its-application-in-mining-waste-purification-medicine-biochemistry-and-chemistry
#18
REVIEW
M Iranmanesh, J Hulliger
The use of strong magnetic field gradients and high magnetic fields generated by permanent magnets or superconducting coils has found applications in many fields such as mining, solid state chemistry, biochemistry and medical research. Lab scale or industrial implementations involve separation of macro- and nanoparticles, cells, proteins, and macromolecules down to small molecules and ions. Most promising are those attempts where the object to be separated is attached to a strong magnetic nanoparticle. Here, all kinds of specific affinity interactions are used to attach magnetic carrier particles to mainly objects of biological interest...
July 21, 2017: Chemical Society Reviews
https://www.readbyqxmd.com/read/28729020/purification-of-cell-culture-derived-influenza-a-virus-via-continuous-anion-exchange-chromatography-on-monoliths
#19
Laura M Fischer, Michael W Wolff, Udo Reichl
The continuously increasing demand for potent and safe vaccines and the intensifying economic pressure on health care systems underlines the need for further optimization of vaccine manufacturing. Here, we focus on downstream processing of human influenza vaccines, investigating the purification of serum-free cell culture-derived influenza virus (A/PR/8/34 H1N1) using continuous chromatography. Therefore, quaternary amine anion exchange monoliths (CIM® QA) were characterized for their capacity to capture virus particles from animal cells cultivated in different media and their ability to separate virions from contaminating host cell proteins and DNA...
July 17, 2017: Vaccine
https://www.readbyqxmd.com/read/28728943/production-and-biophysical-characterization-of-a-mini-membrane-protein-ost4v23d-a-functionally-important-mutant-of-yeast-oligosaccharyltransferase-subunit-ost4p
#20
Bharat Chaudhary, Suman Mazumder, Smita Mohanty
N-linked glycosylation of proteins is an essential and highly conserved co- and post-translational protein modification reaction that occurs in all eukaryotes. Oligosaccharyltransferase (OST), a multi-subunit membrane-associated enzyme complex, carries out this reaction. In the central reaction, a carbohydrate group is transferred to the side chain of a consensus asparagine residue in the newly synthesized protein. Genetic defects in humans cause a series of disorders known as congenital disorders of glycosylation (CDG) that include mental retardation, developmental delay, hypoglycemia etc...
July 17, 2017: Protein Expression and Purification
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