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membrane protein purification

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https://www.readbyqxmd.com/read/29767339/simultaneous-decolorization-and-deproteinization-of-%C3%AE-%C3%AF-dodecanedioic-acid-fermentation-broth-by-integrated-ultrafiltration-and-adsorption-treatments
#1
Weifeng Cao, Yujue Wang, Jianquan Luo, Junxiang Yin, Yinhua Wan
α,ω-Dicarboxylic acids (DC) are versatile chemical intermediates with different chain length. For biosynthesis of DC, to obtain the highly pure product via crystallization, it is required to remove pigments and proteins in fermentation broth. However, a trade-off between decolorization/deproteinization ratio and DC recovery during the purification process was found, which impeded DC production by fermentation. When ultrafiltration (UF) was applied to treat α,ω-dodecanedioic acid (DC12 ) broth, 93.4% of DC12 recovery, 80...
May 16, 2018: Bioprocess and Biosystems Engineering
https://www.readbyqxmd.com/read/29751611/purification-and-characterization-of-recombinant-botulinum-neurotoxin-serotype-fa-also-known-as-serotype-h
#2
Gavin Hackett, Kevin Moore, David Burgin, Fraser Hornby, Bryony Gray, Mark Elliott, Imran Mir, Matthew Beard
We have purified and characterized recombinant botulinum neurotoxin serotype FA (BoNT/FA). This protein has also been named as a new serotype (serotype H), but the classification has been controversial. A lack of well-characterized, highly pure material has been a roadblock to study. Here we report purification and characterization of enzymatically active, and of inactive nontoxic, recombinant forms of BoNT/FA as tractable alternatives to purifying this neurotoxin from native Clostridium botulinum . BoNT/FA cleaves the same intracellular target proteins as BoNT/F1 and other F serotype BoNTs; the intracellular targets are vesicle associated membrane proteins (VAMP) 1, 2 and 3...
May 11, 2018: Toxins
https://www.readbyqxmd.com/read/29744292/isolation-of-native-plasma-membrane-h-atpase-pma1p-in-both-the-active-and-basal-activation-states
#3
Jesper Torbøl Pedersen, Tamara Kanashova, Gunnar Dittmar, Michael Palmgren
The yeast plasma membrane H+ -ATPase Pma1p is a P-type ATPase that energizes the yeast plasma membrane. Pma1p exists in two activation states: an autoinhibited basal state and an activated state. Here we show that functional and stable Pma1p can be purified in native form and reconstituted in artificial liposomes without altering its activation state. Acetylated tubulin has previously been reported to maintain Pma1p in the basal state but, as this protein was absent from the purified preparations, it cannot be an essential component of the autoinhibitory mechanism...
May 2018: FEBS Open Bio
https://www.readbyqxmd.com/read/29741359/rapid-water-permeation-through-carbon-nanomembranes-with-sub-nanometer-channels
#4
Yang Yang, Petr Dementyev, Niklas Biere, Daniel Emmrich, Patrick Stohmann, Riko Korzetz, Xianghui Zhang, André Beyer, Sascha Koch, Dario Anselmetti, Armin Gölzhäuser
The provision of clean water is a global challenge, and membrane filtration is a key technology to address it. Conventional filtration membranes are constrained by a trade-off between permeance and selectivity. Recently, some nanostructured membranes demonstrated the ability to overcome this limitation by utilizing well-defined carbon nanoconduits that allow a coordinated passage of water molecules. The fabrication of these materials is still very challenging, but their performance inspires research toward nanofabricated membranes...
May 9, 2018: ACS Nano
https://www.readbyqxmd.com/read/29740434/lack-of-evidence-for-a-direct-interaction-of-progranulin-and-tumor-necrosis-factor-receptor-1-and-tumor-necrosis-factor-receptor-2-from-cellular-binding-studies
#5
Isabell Lang, Simone Füllsack, Harald Wajant
Progranulin (PGRN) is a secreted anti-inflammatory protein which can be processed by neutrophil proteases to various granulins. It has been reported that at least a significant portion of the anti-inflammatory effects of PGRN is due to direct high affinity binding to tumor necrosis factor receptor-1 (TNFR1) and TNFR2 and inhibition of tumor necrosis factor (TNF)-induced TNFR1/2 signaling. Two studies failed to reproduce the interaction of TNFR1 and TNFR2 with PGRN, but follow up reports speculated that this was due to varying experimental circumstances and/or the use of PGRN from different sources...
2018: Frontiers in Immunology
https://www.readbyqxmd.com/read/29739854/from-micelles-to-bicelles-effect-of-the-membrane-on-particulate-methane-monooxygenase-activity
#6
Soo Y Ro, Matthew O Ross, Yue Wen Deng, Sharon Batelu, Thomas J Lawton, Joseph D Hurley, Timothy L Stemmler, Brian M Hoffman, Amy C Rosenzweig
Particulate methane monooxygenase (pMMO) is a copper-dependent, integral membrane metalloenzyme that converts methane to methanol in methanotrophic bacteria. Studies of isolated pMMO have been hindered by loss of enzymatic activity upon its removal from the native membrane. To characterize pMMO in a membrane-like environment, we reconstituted pMMOs from Methylococcus ( Mcc. ) capsulatus (Bath) and Methylomicrobium ( Mm. ) alcaliphilum 20Z into bicelles. Reconstitution into bicelles recovers methane oxidation activity lost upon detergent solubilization and purification without substantial alterations to copper content or copper electronic structure as observed by electron paramagnetic resonance (EPR) spectroscopy...
May 8, 2018: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29738963/purification-of-r-phycoerythrin-from-gracilaria-lemaneiformis-by-centrifugal-precipitation-chromatography
#7
Dongyu Gu, Rodrigo Lazo-Portugal, Chen Fang, Zhantong Wang, Ying Ma, Martha Knight, Yoichiro Ito
Centrifugal precipitation chromatography (CpC) is a powerful chromatographic technique invented in the year 2000 but so far very little applied. The method combines dialysis, counter-current and salting out processes. The separation rotor consists of two identical spiral channels separated by a dialysis membrane (6-8 K MW cut-off) in which the upper channel is eluted with an ammonium sulfate gradient and the lower channel with water, and the mixtures are separated according to their solubility in ammonium sulfate as a chromatographic technique...
May 1, 2018: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
https://www.readbyqxmd.com/read/29738690/recombinant-production-reconstruction-in-lipid-protein-nanodiscs-and-electron-microscopy-of-full-length-%C3%AE-subunit-of-human-potassium-channel-kv7-1
#8
Z O Shenkarev, M G Karlova, D S Kulbatskii, M P Kirpichnikov, E N Lyukmanova, O S Sokolova
Voltage-gated potassium channel Kv7.1 plays an important role in the excitability of cardiac muscle. The α-subunit of Kv7.1 (KCNQ1) is the main structural element of this channel. Tetramerization of KCNQ1 in the membrane results in formation of an ion channel, which comprises a pore and four voltage-sensing domains. Mutations in the human KCNQ1 gene are one of the major causes of inherited arrhythmias, long QT syndrome in particular. The construct encoding full-length human KCNQ1 protein was synthesized in this work, and an expression system in the Pichia pastoris yeast cells was developed...
May 2018: Biochemistry. Biokhimii︠a︡
https://www.readbyqxmd.com/read/29738525/optimizing-exosomal-rna-isolation-for-rna-seq-analyses-of-archival-sera-specimens
#9
Emily N Prendergast, Marcos Abraão de Souza Fonseca, Felipe Segato Dezem, Jenny Lester, Beth Y Karlan, Houtan Noushmehr, Xianzhi Lin, Kate Lawrenson
Exosomes are endosome-derived membrane vesicles that contain proteins, lipids, and nucleic acids. The exosomal transcriptome mediates intercellular communication, and represents an understudied reservoir of novel biomarkers for human diseases. Next-generation sequencing enables complex quantitative characterization of exosomal RNAs from diverse sources. However, detailed protocols describing exosome purification for preparation of exosomal RNA-sequence (RNA-Seq) libraries are lacking. Here we compared methods for isolation of exosomes and extraction of exosomal RNA from human cell-free serum, as well as strategies for attaining equal representation of samples within pooled RNA-Seq libraries...
2018: PloS One
https://www.readbyqxmd.com/read/29736150/diagnostic-potential-of-brucella-melitensis-rev1-native-omp28-precursor-in-human-brucellosis
#10
Ismail Koyuncu, Abdurrahman Kocyigit, Ahmet Ozer, Sahabettin Selek, Adnan Kirmit, Hasan Karsen
Serologic tests for brucellosis aim to detect antibodies produced against membranous lipopolysaccharide of bacteria. Diagnostic use of this method is limited due to false positiveness. This study evaluates an alternative antigen to lipopolysaccharides (LPS), outer membrane 28-precursor-protein, of Brucella melitensis Rev1 for its diagnostic value. Omp28 precursor of B. melitensis Rev1 was cloned, expressed, and purified. 6-His and sumo epitope tags were used to tag the protein at N-termini. Omp28 gene was amplified based on the ORF sequence and cloned into a pETSUMO vector...
2018: Central-European Journal of Immunology
https://www.readbyqxmd.com/read/29729432/quantitative-proteomics-and-biochemical-analyses-reveal-the-role-of-endoplasmin-in-the-regulation-of-the-expression-and-secretion-of-a-disintegrin-and-metalloproteinase-12
#11
Lipeng Xiong, Wenwen Yan, Emmanuel Zubia, Yanqing Zhou, Yang Zhang, Qianqian Duan, Mahesh Narayan, Guoqiang Xu
A Disintegrin And Metalloproteinase 12 (ADAM12) is highly expressed in multiple cancers such as breast and cervical cancers and its high expression reduces the overall patient survival rate. ADAM12 has two major splicing variants, the long membrane-anchored form ADAM12L and the short secreted form ADAM12S. However, how they are regulated and whether they are modulated similarly or differently in cells are not clear. Here, we use affinity purification and mass spectrometry to identify the ADAM12S-interacting proteins...
May 2, 2018: Journal of Proteomics
https://www.readbyqxmd.com/read/29712826/catalytic-amino-acid-production-from-biomass-derived-intermediates
#12
Weiping Deng, Yunzhu Wang, Sui Zhang, Krishna M Gupta, Max J Hülsey, Hiroyuki Asakura, Lingmei Liu, Yu Han, Eric M Karp, Gregg T Beckham, Paul J Dyson, Jianwen Jiang, Tsunehiro Tanaka, Ye Wang, Ning Yan
Amino acids are the building blocks for protein biosynthesis and find use in myriad industrial applications including in food for humans, in animal feed, and as precursors for bio-based plastics, among others. However, the development of efficient chemical methods to convert abundant and renewable feedstocks into amino acids has been largely unsuccessful to date. To that end, here we report a heterogeneous catalyst that directly transforms lignocellulosic biomass-derived α-hydroxyl acids into α-amino acids, including alanine, leucine, valine, aspartic acid, and phenylalanine in high yields...
April 30, 2018: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29709556/goldclip-gel-omitted-ligation-dependent-clip
#13
Jiaqi Gu, Ming Wang, Yang Yang, Ding Qiu, Yiqun Zhang, Jinbiao Ma, Yu Zhou, Gregory J Hannon, Yang Yu
Protein-RNA interaction networks are essential to understand gene regulation control. Identifying binding sites of RNA-binding proteins (RBPs) by the UV-crosslinking and immunoprecipitation (CLIP) represents one of the most powerful methods to map protein-RNA interactions in vivo. However, the traditional CLIP protocol is technically challenging, which requires radioactive labeling and suffers from material loss during PAGE-membrane transfer procedures. Here we introduce a super-efficient CLIP method (GoldCLIP) that omits all gel purification steps...
April 27, 2018: Genomics, Proteomics & Bioinformatics
https://www.readbyqxmd.com/read/29698455/isolation-of-mitochondria-from-saccharomyces-cerevisiae-using-magnetic-bead-affinity-purification
#14
Pin-Chao Liao, Istvan R Boldogh, Stephanie E Siegmund, Zachary Freyberg, Liza A Pon
Isolated mitochondria are widely used to study the function of the organelle. Typically, mitochondria are prepared using differential centrifugation alone or in conjunction with density gradient ultracentrifugation. However, mitochondria isolated using differential centrifugation contain membrane or organelle contaminants, and further purification of crude mitochondria by density gradient ultracentrifugation requires large amounts of starting material, and is time-consuming. Mitochondria have also been isolated by irreversible binding to antibody-coated magnetic beads...
2018: PloS One
https://www.readbyqxmd.com/read/29678587/folding-and-stabilizing-membrane-proteins-in-amphipol-a8-35
#15
REVIEW
Christel Le Bon, Anaïs Marconnet, Sandrine Masscheleyn, Jean-Luc Popot, Manuela Zoonens
Membrane proteins (MPs) are important pharmacological targets because of their involvement in many essential cellular processes whose dysfunction can lead to a large variety of diseases. A detailed knowledge of the structure of MPs and the molecular mechanisms of their activity is essential to the design of new therapeutic agents. However, studying MPs in vitro is challenging, because it generally implies their overexpression under a functional form, followed by their extraction from membranes and purification...
April 17, 2018: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/29658698/hydrophobic-anti-fouling-electrospun-mats-from-zwitterionic-amphiphilic-copolymers
#16
Sefika Ozcan, Papatya Kaner, David Thomas, Peggy Cebe, Ayse Asatekin
A porous material that is both hydrophobic and fouling resistant is needed in many applications, such as water purification by membrane distillation. In this work, we take a novel approach to fabricating such membranes. Using the zwitterionic amphiphilic copolymer poly(trifluoroethyl methacrylate-random-sulfobetaine methacrylate) (PTFEMA-r-SBMA), we electrospin non-woven, porous membranes that combine high hydrophobicity with resistance to protein adsorption. By changing electrospinning parameters and solution composition, membranes can be prepared with a wide range of fiber morphologies including beaded, bead-free, wrinkly, and ribbon-like fibers, with diameters ranging between ~150 nm and 1...
April 16, 2018: ACS Applied Materials & Interfaces
https://www.readbyqxmd.com/read/29651698/mitogenic-activity-of-artocarpus-lingnanensis-lectin-and-its-apoptosis-induction-in-jurkat-t-cells
#17
Linjie Zeng, Lu Li, Qiyan Zeng, Yong Deng, Lijun Yin, Liejun Liao
Lectins are a class of carbohydrate-binding proteins or glycoproteins and used in the purification and characterization of glycoproteins according to their specificity to carbohydrates. In the present study, the mitogenic activity of Artocarpus lingnanensis lectin (ALL) and its apoptosis induction in Jurkat T cells were explored. MTT assay revealed strong mitogenic potential of ALL. Meanwhile, the anti-cancer activity of ALL was also explored using the human leukemic Jurkat T cell line. ALL exhibited strong binding affinity (97%) to the cell membrane, which could be effectively inhibited by N-acetyl-D-galactosaminide (NAD)...
April 12, 2018: Journal of Natural Medicines
https://www.readbyqxmd.com/read/29625104/structural-requirement-of-the-hydrophobic-region-of-the-bordetella-pertussis-cyaa-hemolysin-for-functional-association-with-cyac-acyltransferase-in-toxin-acylation
#18
Veerada Raksanoh, Panchika Prangkio, Chompounoot Imtong, Niramon Thamwiriyasati, Kittipong Suvarnapunya, Lalida Shank, Chanan Angsuthanasombat
Previously, we demonstrated that the ∼130-kDa CyaA-hemolysin (CyaA-Hly, Met482 -Arg1706 ) from Bordetella pertussis was palmitoylated at Lys983 when co-expressed with CyaC-acyltransferase in Escherichia coli, and thus activated its hemolytic activity. Here, further investigation on a possible requirement of the N-terminal hydrophobic region (HP, Met482 -Leu750 ) for toxin acylation was performed. The ∼100-kDa RTX (Repeat-in-ToXin) fragment (CyaA-RTX, Ala751 -Arg1706 ) containing the K983 -acylation region (AR, Ala751 -Gln1000 ), but lacking HP, was co-produced with CyaC in E...
April 3, 2018: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/29618733/use-of-soluble-extracellular-regions-of-mmpl-serom-as-vaccines-for-tuberculosis
#19
Emily J Strong, Nicholas P West
The current vaccine for tuberculosis (TB) is a live attenuated strain of Mycobacterium bovis (BCG) and while effective at reducing the potential for disseminated TB in young children its disease protection rates in adults is highly variable while it confers little protection against latent TB. With these limitations a new vaccine is desperately needed. We investigated the efficacy of three members of the mycobacterial membrane protein Large (MmpL) family as potential subunit vaccines for TB. MmpLs are large, multifunctional integral membrane proteins, and as such are recalcitrant to purification...
April 4, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29608964/purification-of-membrane-proteins-free-from-conventional-detergents-sma-new-polymers-new-opportunities-and-new-insights
#20
REVIEW
Zoe Stroud, Stephen C L Hall, Tim R Dafforn
Membrane proteins remain a somewhat enigmatic group of biomolecules. On the one hand they mediate some of the most important processes in biology with molecular mechanisms that are often elegantly complex. On the other hand they are exceptionally challenging to produce, making studies of membrane protein structure and function among the most difficult projects undertaken by biochemists. The central issue with studies of a membrane protein has been the need to extract them from their native lipid environment before purification and production of a homogenous sample...
March 30, 2018: Methods: a Companion to Methods in Enzymology
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