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Protein expression and purification

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https://www.readbyqxmd.com/read/28648677/cell-type-specific-transcriptome-profiling-in-c-elegans-using-the-translating-ribosome-affinity-purification-technique
#1
Xicotencatl Gracida, John A Calarco
Organs and specific cell types execute specialized functions in multicellular organisms, in large part through customized gene expression signatures. Thus, profiling the transcriptomes of specific cell and tissue types remains an important tool for understanding how cells become specialized. Methodological approaches to detect gene expression differences have utilized samples from whole animals, dissected tissues, and more recently single cells. Despite these advances, there is still a challenge and a need in most laboratories to implement less invasive yet powerful cell-type specific transcriptome profiling methods...
June 22, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28648085/specifying-rna-binding-regions-in-proteins-by-peptide-cross-linking-and-affinity-purification
#2
Meeli Mullari, David Lyon, Lars Juhl Jensen, Michael L Nielsen
RNA-binding proteins (RBPs) allow cells to carry out pre-RNA processing and post-transcriptional regulation of gene expression, with aberrations in RBP functions linked to many diseases including neurological disorders and cancer. Human cells encode thousands of RNA-binding proteins with unique RNA-binding properties. These properties are regulated through modularity of a large variety of RNA-binding domains, rendering RNA-protein interactions difficult to study. Recently, the introduction of proteomics methods has provided novel insights into RNA-binding proteins at a systems level...
June 25, 2017: Journal of Proteome Research
https://www.readbyqxmd.com/read/28646324/expression-purification-and-characterization-of-gmz2-10c-a-complex-disulphide-bonded-fusion-protein-vaccine-candidate-against-the-asexual-and-sexual-life-stages-of-the-malaria-causing-plasmodium-falciparum-parasite
#3
Ulrik H Mistarz, Susheel K Singh, Tam T T N Nguyen, Will Roeffen, Fen Yang, Casper Lissau, Søren M Madsen, Astrid Vrang, Régis W Tiendrebeogo, Ikhlaq H Kana, Robert W Sauerwein, Michael Theisen, Kasper D Rand
PURPOSE: Production and characterization of a chimeric fusion protein (GMZ2'.10C) which combines epitopes of key malaria parasite antigens: glutamate-rich protein (GLURP), merozoite surface protein 3 (MSP3), and the highly disulphide bonded Pfs48/45 (10C). GMZ2'.10C is a potential candidate for a multi-stage malaria vaccine that targets both transmission and asexual life-cycle stages of the parasite. METHODS: GMZ2'.10C was produced in Lactococcus lactis and purified using either an immunoaffinity purification (IP) or a conventional purification (CP) method...
June 23, 2017: Pharmaceutical Research
https://www.readbyqxmd.com/read/28645932/a-functional-nav1-7-navab-chimera-with-a-reconstituted-high-affinity-protx-ii-binding-site
#4
Ramkumar Rajamani, Sophie Wu, Iyoncy Rodrigo, Mian Gao, Simon Low, Lisa Megson, David Wensel, Rick Pieschl, Debra Post-Munson, John Watson, David R Langley, Michael Ahlijanian, Linda Bristow, James Herrington
NaV1.7 is genetically implicated in human pain perception. Rare gain of function mutations in NaV1.7 lead to spontaneous pain in humans whereas loss of function mutations result in congenital insensitivity to pain (CIP). Hence, agents that specifically modulate the function of NaV1.7 have the potential to yield novel therapeutics to treat pain. The complexity of the channel and the challenges to generate recombinant cell lines with high NaV1.7 expression have led to a templated target strategy approach employing chimeras with the bacterial channel, NavAb...
June 23, 2017: Molecular Pharmacology
https://www.readbyqxmd.com/read/28644752/expression-and-purification-of-swine-rag2-in-e-coli-for-production-of-porcine-rag2-polyclonal-antibodies
#5
Yu-Bei Jin, Wen-Tao Yang, Ke-Yan Huang, Hong-Liang Chen, Seria-Masole Shonyela, Jing Liu, Qiong Liu, Bo Feng, You Zhou, Shu-Li Zhi, Yan-Long Jiang, Jian-Zhong Wang, Hai-Bin Huang, Chun-Wei Shi, Gui-Lian Yang, Chun-Feng Wang
Recombination activating gene 2 (RAG2) is necessary for immature B cell differentiation. Antibodies to human and rabbit RAG2 are currently commercially available, but antibodies to swine RAG remain unavailable to date. In this study, the swine RAG2 genes sequence was synthesized and then cloned into a pET-28a vector. The recombinant fusion protein was successfully expressed in E. coli, purified through nickel column chromatography, and further digested with Tobacco Etch Virus protease. The cleaved protein was purified by molecular-exclusion chromatography and named pRAG2...
June 23, 2017: Bioscience, Biotechnology, and Biochemistry
https://www.readbyqxmd.com/read/28642005/expression-purification-and-crystallization-of-type-1-isocitrate-dehydrogenase-from-trypanosoma-brucei
#6
Xinying Wang, Daniel Ken Inaoka, Tomoo Shiba, Emmanuel Oluwadare Balogun, Stefan Allmann, Yoh-Ichi Watanabe, Michael Boshart, Kiyoshi Kita, Shigeharu Harada
Isocitrate dehydrogenases (IDHs) are metabolic enzymes that catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate. Depending on the electron acceptor and subcellular localization, these enzymes are classified as NADP(+)-dependent IDH1 in the cytosol or peroxisomes, NADP(+)-dependent IDH2 and NAD(+)-dependent IDH3 in mitochondria. Trypanosoma brucei is a protozoan parasite that causes African sleeping sickness in humans and Nagana disease in animals. Here, for the first time, a putative glycosomal T...
June 19, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28641610/-biological-characteristics-of-microvesicles-secreted-by-human-peripheral-blood-hematopoietic-stem-cells
#7
Chen Liang, Jun-Hui Wang, Lei Deng, Lu Wang, Yi Wang, Ya-Jing Huang, Tie-Qiang Liu, Bo Cai, Hong-Li Zuo, Qi-Yun Sun, Jian-Hui Qiao, Chang-Lin Yu, Kai-Xun Hu, Hui-Sheng Ai, Mei Guo
OBJECTIVE: To investigate the effects of microvesicles(MV) isolated from human peripheral blood hematopoietic stem cells(PB-HSC) on immune regulation and hematopoiesis. METHODS: PB-HSCs were separated by density-gradient centrifugation and cultrued. The supernatants of PB-HSC at 48 h were harvested for isolation and purification of MV by using ultracentrifugation. The electron microscopy was used to observe the morphology of MV. The protein level in MV was quantified through bicinchoninic acid(BCA) protein assay...
June 2017: Zhongguo Shi Yan Xue Ye Xue za Zhi
https://www.readbyqxmd.com/read/28640955/in-solution-antibody-harvesting-with-a-plant-produced-hydrophobin-protein-a-fusion
#8
Katri Kurppa, Lauri Reuter, Anneli Ritala, Markus Linder, Jussi Joensuu
Purification is a bottleneck and a major cost factor in the production of antibodies. We set out to engineer a bi-functional fusion protein from two building blocks, Protein A and a hydrophobin, aiming at low-cost and scalable antibody capturing in solutions. Immunoglobulin-binding Protein A is widely used in affinity-based purification. The hydrophobin fusion tag, on the other hand, has been shown to enable purification by two-phase separation. Protein A was fused to two different hydrophobin tags, HFBI or II, and expressed transiently in Nicotiana benthamiana...
June 22, 2017: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/28640945/production-of-human-vitronectin-in-nicotiana-benthamiana-using-the-inpact-hyper-expression-platform
#9
Benjamin Dugdale, Maiko Kato, Pradeep Deo, Manuel Plan, Mark Harrison, Robyn Lloyd, Terry Walsh, Robert Harding, James Dale
Human vitronectin (hVN) is a glycoprotein that functions as a cell adhesion molecule and a regulator of coagulation in blood plasma and the extracellular matrix. In vitro, hVN is added to serum-free media in order to promote the adhesion of animal cells to tissue culture surfaces and the proliferation of undifferentiated stem cells. Here, we report the production of hVN in Nicotiana benthamiana using the inducible In Plant Activation (INPACT) hyper-expression platform. N. benthamiana plants were transformed with an INPACT expression cassette encoding hVN, and both the Tobacco yellow dwarf virus Rep/RepA activator and Tomato bushy stunt virus p19 gene under the transcriptional control of the ethanol-inducible AlcR:alcA gene switch...
June 22, 2017: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/28634775/hydrophilic-acylated-surface-protein-a-haspa-of-leishmania-donovani-expression-purification-and-biophysico-chemical-characterization
#10
Manoj Kumar, Kishu Ranjan, Vijay Singh, Chandramani Pathak, Anju Pappachan, Desh Deepak Singh
Hydrophilic acylated surface proteins (HASPs) are acidic surface proteins which get localized on the surface of Leishmania parasite during infective stages through a "non-classical" pathway. In this study, we report the heterologous expression and purification of Leishmania donovani HASPA (r-LdHASPA) in E. coli system and its partial characterization. The structural aspects of the purified protein were analyzed using CD spectroscopy and modeling studies which indicate that r-LdHASPA consists of random coils...
June 21, 2017: Protein Journal
https://www.readbyqxmd.com/read/28631997/expression-and-purification-of-a-functional-porcine-soluble-triggering-receptor-expressed-on-myeloid-cells-1
#11
Hui Li, Shuangshuang Guo, Leyan Yan, Chunhua Meng, Yiyi Hu, Kongwang He, Zhendan Shi
Triggering receptor expressed on myeloid cells 1 (TREM-1) plays a vital role in the pathogen-triggered amplification loop required for proinflammatory responses. Blockade of TREM-1 signaling may inhibit expansion of sepsis and prolong survival of animals. In the present study, the gene of porcine soluble TREM-1 was cloned and expressed in E. coli. After purification, the bioactivity of recombinant porcine soluble TREM-1 was tested in vitro on porcine alveolar macrophages. The results showed that supplementation with the recombinant porcine sTREM-1 protein rapidly and dose-dependently attenuated the upregulation of cytokines (IL-1β, IL-2, IL-4, IL-8, IL-10, IL-12, IL-16, IL-18, and TNF-α) caused by LPS stimulation in the cultured porcine alveolar macrophages...
February 10, 2017: Animal Biotechnology
https://www.readbyqxmd.com/read/28630382/immunodiagnostic-value-of-echinococcus-granulosus-recombinant-b8-1-subunit-of-antigen-b
#12
Amir Savardashtaki, Bahador Sarkari, Farzane Arianfar, Zohreh Mostafavi-Pour
BACKGROUND: Cystic echinococcosis (CE), as a chronic parasitic disease, is a major health problem in many countries. The performance of the currently available serodiagnostic tests for the diagnosis of CE is unsatisfactory. OBJECTIVE: The current study aimed at sub-cloning a gene, encoding the B8/1 subunit of antigen B (AgB) from Echinococcus granulosus, using gene optimization for the immunodiagnosis of human CE. METHODS: The coding sequence for AgB8/1 subunit of Echinococcus granulosus was selected from GenBank and was gene-optimized...
June 2017: Iranian Journal of Immunology: IJI
https://www.readbyqxmd.com/read/28628608/tcp4-dependent-induction-of-constans-transcription-requires-gigantea-in-photoperiodic-flowering-in-arabidopsis
#13
Akane Kubota, Shogo Ito, Jae Sung Shim, Richard S Johnson, Yong Hun Song, Ghislain Breton, Greg S Goralogia, Michael S Kwon, Dianne Laboy Cintrón, Tomotsugu Koyama, Masaru Ohme-Takagi, Jose L Pruneda-Paz, Steve A Kay, Michael J MacCoss, Takato Imaizumi
Photoperiod is one of the most reliable environmental cues for plants to regulate flowering timing. In Arabidopsis thaliana, CONSTANS (CO) transcription factor plays a central role in regulating photoperiodic flowering. In contrast to posttranslational regulation of CO protein, still little was known about CO transcriptional regulation. Here we show that the CINCINNATA (CIN) clade of class II TEOSINTE BRANCHED 1/ CYCLOIDEA/ PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR (TCP) proteins act as CO activators. Our yeast one-hybrid analysis revealed that class II CIN-TCPs, including TCP4, bind to the CO promoter...
June 19, 2017: PLoS Genetics
https://www.readbyqxmd.com/read/28628207/engineered-polymerases-with-altered-substrate-specificity-expression-and-purification
#14
Ali Nikoomanzar, Matthew R Dunn, John C Chaput
Polymerase engineering is making it possible to synthesize xeno-nucleic acid polymers (XNAs) with diverse backbone structures and chemical functionality. The ability to copy genetic information back and forth between DNA and XNA has led to a new field of science known as synthetic genetics, which aims to study the genetic concepts of heredity and evolution in artificial genetic polymers. Since many of the polymerases needed to synthesize XNA polymers are not available commercially, researchers must express and purify these enzymes as recombinant proteins from E...
June 19, 2017: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/28627567/dissecting-binding-of-a-%C3%AE-barrel-membrane-protein-by-phage-display
#15
Luz M Meneghini, Sarvind Tripathi, Marcus A Woodworth, Sudipta Majumdar, Thomas L Poulos, Gregory A Weiss
Membrane proteins (MPs) constitute a third of all proteomes, and contribute to a myriad of cellular functions including intercellular communication, nutrient transport and energy generation. For example, TonB-dependent transporters (TBDTs) in the outer membrane of Gram-negative bacteria play an essential role transporting iron and other nutrients into the bacterial cell. The inherently hydrophobic surfaces of MPs complicates protein expression, purification, and characterization. Thus, dissecting the functional contributions of individual amino acids or structural features through mutagenesis can be a challenging ordeal...
June 19, 2017: Molecular BioSystems
https://www.readbyqxmd.com/read/28625912/isolation-of-recombinant-human-untagged-glyceraldehyde-3-phosphate-dehydrogenase-from-e-%C3%A2-coli-producer-strain
#16
K V Barinova, M A Eldarov, E V Khomyakova, V I Muronetz, E V Schmalhausen
The goal of the present work was expression of human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) without additional tag constructions in E. coli cells and elaboration of the procedure for purification of untagged hGAPDH from the extract of the producer cells. We present a simple method for purification of untagged hGAPDH including ammonium sulfate fractionation and gel filtration on a G-100 Sephadex column. The method allows isolation of 2 mg of pure hGAPDH from 600 ml of cell culture (7 g of the cell biomass)...
June 15, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28625911/white-shrimp-litopenaeus-vannamei-recombinant-lactate-dehydrogenase-biochemical-and-kinetic-characterization
#17
Ambar A Fregoso-Peñuñuri, Elisa M Valenzuela-Soto, Ciria G Figueroa-Soto, Alma B Peregrino-Uriarte, Manuel Ochoa-Valdez, Lilia Leyva-Carrillo, Gloria Yepiz-Plascencia
Shrimp lactate dehydrogenase (LDH) is induced in response to environmental hypoxia. Two protein subunits deduced from different transcripts of the LDH gene from the shrimp Litopenaeus vannamei (LDHvan-1 and LDHvan-2) were identified. These subunits are expressed by alternative splicing. Since both subunits are expressed in most tissues, the purification of the enzyme from the shrimp will likely produce hetero LDH containing both subunits. Therefore, the aim of this study was to overexpress, purify and characterize only one subunit as a recombinant protein, the LDHvan-2...
June 15, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28624997/expression-and-purification-of-cytochrome-p450-55b1-from-chlamydomonas-reinhardtii-and-its-application-in-nitric-oxide-biosensing
#18
Bin Gong, Xiaosheng Liang, Yong Li, Qian Xiao, Panchun Yang, Yunhua Wu
Cytochrome P450 55B1 from Chlamydomonas reinhardtii is reported to function as a nitric oxide reductase (NOR). Here, we expressed the cytochrome P450 55B1 gene with an HIS-tag in E scherichia coli using a pET28a vector. The native protein was produced at a level of 1.59 μmol/g of total protein, with approximately 85% of the P450 being soluble. The CYP55B1 protein was characterized spectrally and purified by a HIS-trap column. This procedure allowed recovery of 45% of the expressed protein and CYP55B1 with a specific content of 0...
June 17, 2017: Applied Biochemistry and Biotechnology
https://www.readbyqxmd.com/read/28624493/expression-of-the-c-terminal-domain-of-human-apolipoprotein-a-i-using-a-chimeric-apolipoprotein
#19
Daniel E Sallee, James V C Horn, Lukas A Fuentes, Paul M M Weers
Human apolipoprotein A-I (apoA-I) is the most abundant protein in high-density lipoprotein, an anti-atherogenic lipid-protein complex responsible for reverse cholesterol transport. The protein is composed of an N-terminal helix bundle domain, and a small C-terminal (CT) domain. To facilitate study of CT-apoA-I, a novel strategy was employed to produce this small domain in a bacterial expression system. A protein construct was designed of insect apolipophorin III (apoLp-III) and residues 179-243 of apoA-I, with a unique methionine residue positioned between the two proteins and an N-terminal His-tag to facilitate purification...
June 15, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28623136/arabidopsis-inositol-polyphosphate-kinase-atipk2%C3%AE-is-phosphorylated-by-cpk4-and-positively-modulates-aba-signaling
#20
Peng Wang, Qiaofeng Yang, Sihong Sang, Yao Chen, Yujiao Zhong, Zhaoyun Wei
Arabidopsis inositol polyphosphate kinase 2β (AtIpk2β) has multiple functions in plant development and in responding to abiotic stress. Although some related clues suggested a potential role of AtIpk2β in ABA signaling, the defined evidence was still lack. Here we discovered that a key ABA signaling component calcium-dependent protein kinase 4 (CPK4) can interact with AtIpk2β under ABA treated conditions through affinity purification and mass spectrometry detection. The interaction between CPK4 and AtIpk2β were further confirmed by yeast two hybrid and bimolecular fluorescence complementation assays...
June 13, 2017: Biochemical and Biophysical Research Communications
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