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https://www.readbyqxmd.com/read/29331148/isolation-of-anti-extra-cellular-vesicle-single-domain-antibodies-by-direct-panning-on-vesicle-enriched-fractions
#1
Milica Popovic, Elisa Mazzega, Barbara Toffoletto, Ario de Marco
BACKGROUND: The thorough understanding of the physiological and pathological processes mediated by extracellular vesicles (EVs) is challenged by purification methods which are cumbersome, not reproducible, or insufficient to yield homogeneous material. Chromatography based on both ion-exchange and immune-capture can represent an effective method to improve EV purification and successive analysis. METHODS: Cell culture supernatant was used as a model sample for assessing the capacity of anion-exchange chromatography to separate distinct EV fractions and to isolate nanobodies by direct panning on whole EVs to recover binders specific for the native conformation of EV-surface epitopes and suitable to develop EV immune-capture reagents...
January 13, 2018: Microbial Cell Factories
https://www.readbyqxmd.com/read/29331014/biochemical-characterization-of-recombinant-thermostable-cohnella-sp-a01-%C3%AE-glucanase
#2
Meysam Rezaie, Saeed Aminzadeh, Farid Heidari, Masoud Mashhadi Akbar Boojar, Ali Asghar Karkhane
Background: Typically, non-cellulytic glucanase, including fungi and yeast cell wall hydrolyzing enzymes, are release by some symbiotic fungi and plants during the mycoparasitic fungi attack on plants. These enzymes are known as the defense mechanisms of plants. This study intends to investigate the biochemical properties of β-1,6-glucanase (bg16M) from native thermophilic bacteria, Cohnella A01. Methods: bg16M gene was cloned and expressed in E. coli BL21 (DE3)...
January 13, 2018: Iranian Biomedical Journal
https://www.readbyqxmd.com/read/29330912/high-capacity-protein-a-affinity-chromatography-for-the-fast-quantification-of-antibodies-two-wavelength-detection-expands-linear-range
#3
Peter Satzer, Alois Jungbauer
The high-throughput analysis of antibodies from processes can be enhanced when the linear range is expanded and sample preparation is kept to a minimum. We developed a fast chromatography method based on a hexameric variant of Staphylococcal protein A immobilized on Toyopearl matrix, TSK 5 PW using two wavelengths. A protocol with 5 min runtime and a single-wavelength detection at 280 nm yielded an upper limit of quantification of 2.10 mg/mL and a lower limit of quantification of 0.06 mg/mL. The optimized method with a runtime of 2 min and two-wavelength detection at 280 and 300 nm allowed us to span a valid concentration range of 0...
January 13, 2018: Journal of Separation Science
https://www.readbyqxmd.com/read/29329805/preparative-expression-and-purification-of-a-nacreous-protein-n16-and-testing-its-effect-on-osteoporosis-rat-model
#4
Zhen-Yan Xu, Yu-Ling Liu, Jia-Bi Lin, Ke-Ling Cheng, Yong-Gang Wang, Hong-Liang Yao, Wei-Peng, Hoi-Yan Wu, Wei-Wei Su, Pang-Chui Shaw, Pei-Bo Li
N16, a nacreous protein isolated from Pinctada martensii, is related to nacreous layer formation. Our previous study indicated that N16 showed dual regulatory effects by inducing osteoblast biomineralization as well as inhibiting osteoclast formation. In order to obtain large quantity of N16 for animal experiment and clinical trial, a fermentation and preparative purification method was established. The N16 cDNA was cloned to a BL21(DE3)plysE-pET32a vector and grown in a 20 L fermenter. The medium, temperature, pH and dissolved oxygen (DO) were optimized...
January 9, 2018: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/29326975/identifying-the-substrate-proteins-of-u-box-e3s-e4b-and-chip-by-orthogonal-ubiquitin-transfer
#5
Karan Bhuripanyo, Yiyang Wang, Xianpeng Liu, Li Zhou, Ruochuan Liu, Duc Duong, Bo Zhao, Yingtao Bi, Han Zhou, Geng Chen, Nicholas T Seyfried, Walter J Chazin, Hiroaki Kiyokawa, Jun Yin
E3 ubiquitin (UB) ligases E4B and carboxyl terminus of Hsc70-interacting protein (CHIP) use a common U-box motif to transfer UB from E1 and E2 enzymes to their substrate proteins and regulate diverse cellular processes. To profile their ubiquitination targets in the cell, we used phage display to engineer E2-E4B and E2-CHIP pairs that were free of cross-reactivity with the native UB transfer cascades. We then used the engineered E2-E3 pairs to construct "orthogonal UB transfer (OUT)" cascades so that a mutant UB (xUB) could be exclusively used by the engineered E4B or CHIP to label their substrate proteins...
January 2018: Science Advances
https://www.readbyqxmd.com/read/29326063/efficient-purification-of-a-highly-active-h-subunit-of-tyrosinase-from-agaricus-bisporus
#6
David Lopez-Tejedor, Jose M Palomo
A highly-active tyrosinase (H subunit) isoform has been purified from a commercial crude extract of Agaricus bisporus by a specific, two step-hydrophobic chromatography cascade process based on the differential adsorption of the proteins from the extract to hydrophobic-functionalized supports. At first, commercial, crude tyrosinase from Agaricus bisporus (AbTyr) dissolved in aqueous media was added to octadecyl-Sepabeads matrix at 25 °C. Under these conditions, the support specifically adsorbed a protein with a molecular weight of 47 kDa which showed no tyrosinase activity...
January 8, 2018: Protein Expression and Purification
https://www.readbyqxmd.com/read/29325877/dmcatd-a-cathepsin-d-like-peptidase-of-the-hematophagous-insect-dipetalogaster-maxima-hemiptera-reduviidae-purification-bioinformatic-analyses-and-the-significance-of-its-interaction-with-lipophorin-in-the-internalization-by-developing-oocytes
#7
Jimena Leyria, Leonardo L Fruttero, Rodrigo Ligabue-Braun, Marina S Defferrari, Estela L Arrese, José L Soulages, Beatriz P Settembrini, Celia R Carlini, Lilián E Canavoso
DmCatD, a cathepsin D-like peptidase of the hematophagous insect Dipetalogaster maxima, is synthesized by the fat body and the ovary and functions as yolk protein precursor. Functionally, DmCatD is involved in vitellin proteolysis. In this work, we purified and sequenced DmCatD, performed bioinformatic analyses and investigated the events involved in its targeting and storage in developing oocytes. By ion exchange and gel filtration chromatography, DmCatD was purified from egg homogenates and its identity was confirmed by mass spectrometry...
January 8, 2018: Journal of Insect Physiology
https://www.readbyqxmd.com/read/29323419/evaluation-of-viral-contamination-in-a-baculovirus-expression-system
#8
Chikako Ono, Junki Hirano, Toru Okamoto, Yoshiharu Matsuura
Insect expression systems based on baculovirus are widely used for the generation of recombinant proteins. Here, we evaluated the infectivity of baculoviruses under the physiological stresses of 'freeze- thaw' and sonication, and the baculoviral contamination of recombinant proteins after protein purification. Our findings suggest that Nonidet™ P-40 (NP-40) treatment of baculoviruses completely abolishes their infectivity and that recombinant proteins purified with affinity beads do not include infectious baculoviruses...
January 11, 2018: Microbiology and Immunology
https://www.readbyqxmd.com/read/29322424/expression-and-purification-of-jak1-and-socs1-for-structural-and-biochemical-studies
#9
Nicholas P D Liau, Jeffrey J Babon
Interferon gamma (IFNγ) is a potent inflammatory and immune cytokine. IFNγ signals via the interferon gamma receptor (IFNGR), which is constitutively bound to Janus Kinase (JAK) 1 and JAK2 via its intracellular domain. These two JAK proteins then initiate the inflammatory signaling cascade. The most potent inhibitor of IFNγ signaling is Suppressor of Cytokine Signaling 1 (SOCS1). SOCS1 negatively regulates IFNγ signaling pathway (and other pathways) by directly inhibiting JAKs. Here, we describe a protocol for the recombinant production and purification of the JAK1 kinase domain and its inhibitor SOCS1, for structural and biochemical studies...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29321325/the-identification-and-characterization-of-sindbis-virus-rna-host-protein-interactions
#10
Autumn T LaPointe, Natasha N Gebhart, Megan E Meller, Richard W Hardy, Kevin J Sokoloski
Arthropod-borne viruses, such as the members of genus Alphavirus, are a significant concern to global public health. As obligate intracellular pathogens, RNA viruses must interact with the host cell machinery to establish, and complete, their viral lifecycles. Despite considerable efforts to define the host/pathogen interactions essential for alphaviral replication, an unbiased and inclusive assessment of alphaviral RNA:protein interactions has not been undertaken. Moreover, the biological and molecular importance of these interactions, in the full context of their molecular function as RNA-binding proteins, has not been fully realized...
January 10, 2018: Journal of Virology
https://www.readbyqxmd.com/read/29320724/purification-and-characterization-of-a-novel-high-molecular-weight-alkaline-protease-produced-by-an-endophytic-bacillus-halotolerans-strain-ct2
#11
Gharbi Dorra, Karkouch Ines, Ben Slimene Imen, Coquet Laurent, Azaiez Sana, Olfa Tabbene, Cosette Pascal, Jouenne Thierry, Limam Ferid
A protease-producing strain CT2 isolated from Tunisian potatoes, exhibiting a potent protease activity (prot CT2), was identified as Bacillus halotolerans according to 16S ribosomal DNA sequence analysis. Maximum prot CT2 production was obtained in medium supplemented with bean seed proteins. Proteolytic activity was purified by ammonium sulphate precipitation, Sephacryl S-200 gel filtration and SP-sepharose cation-exchange chromatography. Optimal enzyme activity was reached at pH 9 and temperature of 50 °C...
January 7, 2018: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/29320632/increasing-the-stability-of-recombinant-human-green-cone-pigment
#12
Timothy S Owen, David Salom, Wenyu Sun, Krzysztof Palczewski
Three types of cone cells exist in the human retina, each containing a different pigment responsible for the initial step of photo-transduction. These pigments are distinguished by their specific absorbance maxima, 425 nm (blue), 530 nm (green) and 560 nm (red). Each pigment contains a common chromophore, 11-cis-retinal covalently bound to an opsin protein via a Schiff base. The 11-cis-retinal protonated Schiff base has an absorbance maxima at 440 nm in methanol. Unfortunately, the chemistry allowing the same chromophore interacting with different opsin proteins to tune the absorbance of the resulting pigments to distinct λmax values is poorly understood...
January 10, 2018: Biochemistry
https://www.readbyqxmd.com/read/29318540/identification-of-proteolytic-cleavage-sites-of-epha2-by%C3%A2-membrane-type-1-matrix-metalloproteinase-on-the%C3%A2-surface-of-cancer-cells
#13
Keiji Kikuchi, Hiroko Kozuka-Hata, Masaaki Oyama, Motoharu Seiki, Naohiko Koshikawa
Proteolytic cleavage of membrane proteins can alter their functions depending on the cleavage sites. We recently demonstrated that membrane type 1 matrix metalloproteinase (MT1-MMP ) converts the tumor suppressor EphA2 into an oncogenic signal transducer through EphA2 cleavage. The cleaved EphA2 fragment that remains at the cell surface may be a better target for cancer therapy than intact EphA2. To analyze the cleavage site(s) of EphA2, we purified the fragments from tumor cells expressing MT1-MMP and Myc- and 6× His-tagged EphA2 by two-step affinity purification ...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29318516/cloning-and-expression-of-h-influenzae-49247-iga-protease-in-e-coli
#14
Honglian Wang, Xia Zhong, Jianchun Li, Menglian Zhu, Lu Wang, Xingli Ji, Junming Fan, Li Wang
IgA protease is secreted by various mucosal pathogenic bacteria which can cleave human immunoglobulin A1 (IgA1) in its hinge region. In addition to be considered as a virulence factor, it's reported that IgA protease can also be used for IgA nephropathy (IgAN) treatment. Our previous study identified bacteria H. influenzae 49247 expressed high activity of IgA protease with promised application in IgAN therapy. In this study, we cloned the IgA protease gene of H. influenzae 49247 with degenerate primers. Alignment analysis indicated that H...
January 9, 2018: Molecular Biotechnology
https://www.readbyqxmd.com/read/29317682/piperonylic-acid-stimulates-keratinocyte-growth-and-survival-by-activating-epidermal-growth-factor-receptor-egfr
#15
Dohyun Lee, Jinsun Lim, Kyung-Chul Woo, Kyong-Tai Kim
Epidermal growth factor (EGF) stimulates cell growth, proliferation, and survival. The biological benefits of EGF have been utilized in medical uses for improving wound healing as well as in today's skin cosmetics. EGF has been found in urine, saliva, milk, and plasma, but its efficient isolation remains a difficult task. With technical advances, recombinant protein purification technique has been used for EGF production. However, the recombinant EGF is still expensive and keeping it with stable activity is difficult to be used widely...
January 9, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29314780/hpv-16-targeted-dna-vaccine-expression-the-role-of-purification
#16
Ana M Almeida, Joana Tomás, Patrícia Pereira, João A Queiroz, Fani Sousa, Ângela Sousa
DNA vaccines have come to light in the last decades as an alternative method to prevent many infectious diseases, but they can also be used for the treatment of specific diseases, such as cervical cancer caused by Human Papillomavirus (HPV). This virus produces E6 and E7 oncoproteins, which alter the cell cycle regulation and can interfere with the DNA repairing system. These features can ultimately lead to the progression of cervical cancer, after cell infection by HPV. Thus, the development of a DNA vaccine targeting both proteins arises as an interesting option in the treatment of this pathology...
January 5, 2018: Biotechnology Progress
https://www.readbyqxmd.com/read/29313422/selection-purification-and-characterization-of-a-her2-targeting-soluble-designed-ankyrin-repeat-protein-by-e-coli-surface-display-using-her2-positive-melanoma-cells
#17
Xiaofei Chen, Xiaoxiao Yu, Xiaoda Song, Li Liu, Yuting Yi, Wenbing Yao, Xiangdong Gao
Human epidermal growth factor receptor 2 (HER2) is a powerful target for cancer immune therapy. The development of anti-HER2 monoclonal antibodies targeting different domains of HER2 is quite effective. However, the selection and production of multivalent antibodies are complicated. In this study, a mimivirus-based designed ankyrin repeat protein (DARPin) targeting HER2 was selected from an artificial library by bacteria surface display. The selection was performed on HER2-positive B16BL6/E2 melanoma cells and HER2-nagative cells...
January 9, 2018: Preparative Biochemistry & Biotechnology
https://www.readbyqxmd.com/read/29311713/towards-high-throughput-gpcr-crystallography-in-meso-soaking-of-adenosine-a2a-receptor-crystals
#18
Prakash Rucktooa, Robert K Y Cheng, Elena Segala, Tian Geng, James C Errey, Giles A Brown, Robert M Cooke, Fiona H Marshall, Andrew S Doré
Here we report an efficient method to generate multiple co-structures of the A2A G protein-coupled receptor (GPCR) with small-molecules from a single preparation of a thermostabilised receptor crystallised in Lipidic Cubic Phase (LCP). Receptor crystallisation is achieved following purification using a low affinity "carrier" ligand (theophylline) and crystals are then soaked in solutions containing the desired (higher affinity) compounds. Complete datasets to high resolution can then be collected from single crystals and seven structures are reported here of which three are novel...
January 8, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29310787/thrombospondins-purification-of-human-platelet-thrombospondin-1
#19
John M Sipes, Joanne E Murphy-Ullrich, David D Roberts
Thrombospondins are a family of five secreted proteins that have diverse roles in modulating cellular function. Thrombospondins-1 and 2 were identified as matricellular proteins based on their functional roles combined with their transient appearance or accumulation in extracellular matrix at specific times during development and in response to injury or stress in mature tissues. Thrombospondin-1 is a major component of platelet α-granules, which provides a convenient source for purification of the protein...
2018: Methods in Cell Biology
https://www.readbyqxmd.com/read/29310786/production-and-purification-of-recombinant-human-sparc
#20
Gail Workman, Amy D Bradshaw
The matricellular protein SPARC (secreted protein acidic and rich in cysteine, also known as osteonectin or as BM-40) is a collagen-binding protein with a capacity to induce cell rounding and influence proliferation in cultured cells. In mice that do not express SPARC, fibrillar collagen is reduced in some adult tissues; notably, a reduction in fibrosis is reported in response to fibrotic stimuli in lungs, heart, skin, liver, and in the eye. Recently, mutations in the gene encoding SPARC were found in patients afflicted with osteogenesis imperfecta...
2018: Methods in Cell Biology
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