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membrane protein purification

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https://www.readbyqxmd.com/read/28748478/urine-exosome-isolation-and-characterization
#1
Jonathan M Street, Erik H Koritzinsky, Deonna M Glispie, Peter S T Yuen
Exosomes are nanometer-scale, membrane-enclosed vesicles that can potentially be used to detect nephrotoxicity, and reveal the subsequent response of the kidney. Epithelial cells of every nephron segment can contribute to the urinary exosome population, which is rich in potential biomarkers, including membrane proteins such as transporters and receptors, transcription factors, and microRNAs. These exosomal biomarkers may be up- or downregulated upon nephrotoxicant exposure. Exosome isolation is an area of ongoing research...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28747506/hcv-induces-the-localization-of-lipid-rafts-to-autophagosomes-for-its-rna-replication
#2
Ja Yeon Kim, Linya Wang, Jiyoung Lee, Jing-Hsiung James Ou
Autophagy plays important roles in maintaining cellular homeostasis. It uses double- or multiple- membrane vesicles termed autophagosomes to remove protein aggregates and damaged organelles from the cytoplasm for recycling. Hepatitis C virus (HCV) has been shown to induce autophagy to enhance its own replication. Here we describe a procedure that combines membrane flotation and affinity chromatography for the purification of autophagosomes from cells that harbor a HCV subgenomic RNA replicon. The purified autophagosomes had double- or multiple- membrane structures with a diameter ranging from 200 nm to 600 nm...
July 26, 2017: Journal of Virology
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/28734361/preparation-of-intact-mitochondria-using-free-flow-isoelectric-focusing-with-post-ph-gradient-sample-injection-for-morphological-functional-and-proteomics-studies
#4
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/28732682/characterization-and-purification-of-proteins-suitable-for-the-production-of-antibodies-against-ca-liberibacter-asiaticus
#5
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/28728943/production-and-biophysical-characterization-of-a-mini-membrane-protein-ost4v23d-a-functionally-important-mutant-of-yeast-oligosaccharyltransferase-subunit-ost4p
#6
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
https://www.readbyqxmd.com/read/28724604/a-simple-toolset-to-identify-endogenous-post-translational-modifications-for-a-target-protein-a-snapshot-of-the-egfr-signaling-pathway
#7
Henrick Horita, Andy Law, Soonjin Hong, Kim Middleton
Identification of a novel post-translational modification (PTM) for a target protein, defining its physiologic role, and studying its potential crosstalk with other PTMs is a challenging process. A set of highly sensitive tools termed Signal-Seeker kits was developed, which enables rapid and simple detection of post-translational modifications on any target protein.  The methodology for these tools utilizes affinity purification of modified proteins from a cell or tissue lysate and immunoblot analysis. These tools utilize a single lysis system that is effective at identifying endogenous, dynamic PTM changes, as well as the potential crosstalk between PTMs...
July 19, 2017: Bioscience Reports
https://www.readbyqxmd.com/read/28719071/mesoporous-silica-thin-membranes-with-large-vertical-mesochannels-for-nanosize-based-separation
#8
Yupu Liu, Dengke Shen, Gang Chen, Ahmed A Elzatahry, Manas Pal, Hongwei Zhu, Longlong Wu, Jianjian Lin, Daifallah Al-Dahyan, Wei Li, Dongyuan Zhao
Membrane separation technologies are of great interest in industrial processes such as water purification, gas separation, and materials synthesis. However, commercial filtration membranes have broad pore size distributions, leading to poor size cutoff properties. In this work, mesoporous silica thin membranes with uniform and large vertical mesochannels are synthesized via a simple biphase stratification growth method, which possess an intact structure over centimeter size, ultrathin thickness (≤50 nm), high surface areas (up to 1420 m(2) g(-1) ), and tunable pore sizes from ≈2...
July 18, 2017: Advanced Materials
https://www.readbyqxmd.com/read/28709863/dissociation-and-purification-of-the-endogenous-membrane-bound-vo-complex-from-pichia-pastoris
#9
Sumei Li, Tao Hong, Kun Wang, Yinghong Lu, Min Zhou
Most proteins occur and function in complexes rather than as isolated entities in membranes. In most cases macromolecules with multiple subunits are purified from endogenous sources. In this study, an endogenous membrane-protein complex was obtained from Pichia pastoris, which can be grown at high densities to significantly improve the membrane protein yield. We successfully isolated the membrane-bound Vo complex of V-ATPase from P. pastoris using a fusion FLAG tag attached to the C-terminus of subunit a to generate the vph-tag strain, which was used for dissociation and purification...
July 12, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28702797/purification-and-antibacterial-mechanism-of-fish-borne-bacteriocin-and-its-application-in-shrimp-penaeus-vannamei-for-inhibiting-vibrio-parahaemolyticus
#10
Xinran Lv, Jingfang Du, Yu Jie, Bolin Zhang, Fengling Bai, Hongfei Zhao, Jianrong Li
Vibrio parahaemolyticus: is recognized as the main cause of gastroenteritis associated with consumption of seafood. Bacteriocin-producing Lactobacillus plantarum FGC-12 isolated from golden carp intestine had strong antibacterial activity toward V. parahaemolyticus. The fish-borne bacteriocin was purified by a three-step procedure consisting of ethyl acetate extraction, gel filtration chromatography and high performance liquid chromatography. Its molecular weight was estimated at 4.1 kDa using SDS-PAGE. The fish-borne bacteriocin reached the maximum production at stationary phase after 20 h...
August 2017: World Journal of Microbiology & Biotechnology
https://www.readbyqxmd.com/read/28698826/expression-refolding-and-spectroscopic-characterization-of-fibronectin-type-iii-fniii-homology-domains-derived-from-human-fibronectin-leucine-rich-transmembrane-protein-flrt-1-2-and-3
#11
Lila Yang, Maria Hansen Falkesgaard, Peter Waaben Thulstrup, Peter Schledermann Walmod, Leila Lo Leggio, Kim Krighaar Rasmussen
The fibronectin leucine rich transmembrane (FLRT) protein family consists in humans of 3 proteins, FLRT1, -2, and -3. The FLRT proteins contain two extracellular domains separated by an unstructured linker. The most membrane distal part is a leucine rich repeat (LRR) domain responsible for both cis- and trans-interactions, whereas the membrane proximal part is a fibronectin type III (FnIII) domain responsible for a cis-interaction with members of the fibroblast growth factor receptor 1 (FGFR1) family, which results in FGFR tyrosine kinase activation...
2017: PeerJ
https://www.readbyqxmd.com/read/28697935/continuous-countercurrent-tangential-chromatography-for-mixed-mode-post-capture-operations-in-monoclonal-antibody-purification
#12
Amit K Dutta, Dmitriy Fedorenko, Jasmine Tan, Joseph A Costanzo, David S Kahn, Andrew L Zydney, Oleg Shinkazh
Continuous Countercurrent Tangential Chromatography (CCTC) has been shown to demonstrate significant advantages over column chromatography including higher productivity, lower operational pressure, disposable flow path, and lower resin use. Previous applications of CCTC have been limited to initial capture of monoclonal antibodies (mAb) from clarified cell culture harvest. In this present article, a CCTC system was designed and tested for a post-capture antibody purification step. Mixed mode cation exchange-hydrophobic interaction chromatography resins with two different particle sizes were used to reduce host cell protein (HCP), leached protein A, DNA, and aggregates from a mAb stream after a protein A operation...
June 22, 2017: Journal of Chromatography. A
https://www.readbyqxmd.com/read/28693207/-99m-tc-lhrh-in-tumor-receptor-imaging
#13
Dawei Hao, Lingfei Sun, Xiang Hu, Xiaowen Hao
Detection of gonadotropin-releasing hormone (GnRH) also known as luteinizing hormone-releasing hormone (LHRH) in the relevant tumor tissue and normal tissues and organs in vivo expression was investigated. To examine the method of direct radio labeling of LHRH by (99m)Tc with relatively high radiochemical purity and stability, screening the best labeling conditions, to establish a simple and reliable method of preparation of (99m)Tc-LHRH was undertaken. The detection of radioisotope-labeled LHRH distribution in mice, LHRH receptor imaging for the study and treatment of cancer basis were evaluated...
July 2017: Oncology Letters
https://www.readbyqxmd.com/read/28692650/expression-purification-and-contaminant-detection-for-structural-studies-of-ralstonia-metallidurance-clc-protein-rm1
#14
Priyanka D Abeyrathne, Nikolaus Grigorieff
Single-particle electron cryo-microscopy (cryo-EM) has become a popular method for high-resolution study of the structural and functional properties of proteins. However, sufficient expression and purification of membrane proteins holds many challenges. We describe methods to overcome these obstacles using ClC-rm1, a prokaryotic chloride channel (ClC) family protein from Ralstonia metallidurans, overexpressed in Escherichia coli (E. coli) BL21(DE3) strain. Mass spectrometry and electron microscopy analyses of purified samples revealed multiple contaminants that can obfuscate results of subsequent high-resolution structural analysis...
2017: PloS One
https://www.readbyqxmd.com/read/28691899/aggregation-of-a-hepatitis-c-virus-replicase-module-induced-by-ablation-of-p97-vcp
#15
Zhigang Yi, Zhenghong Yuan
Hijacking host membranes to assemble a membrane-associated viral replicase is a hallmark of almost all positive-strand RNA viruses. However, how the virus co-opts host factors to facilitate this energy-unfavourable process is incompletely understood. In a previous study, using hepatitis C virus (HCV) as a model and employing affinity purification of the viral replicase, we identified a valosin-containing protein (p97/VCP), a member of the ATPases associated with diverse cellular activities (AAA+ ATPase family), as a viral replicase-associated host factor...
July 10, 2017: Journal of General Virology
https://www.readbyqxmd.com/read/28691022/purification-and-characterization-of-an-atpase-gsia-from-salmonella-enterica
#16
Zhongshan Wang, Meng Zhang, Xiaodong Shi, Quanju Xiang
The coding sequence of Salmonella enterica gsiA was cloned and expressed in E. coli. The protein was purified and ATPase activity was characterized by NADH oxidation method. GsiA exhibited optimum activity at 30°C and at pH 8 in Tris/HCl buffer. GsiA protein was stable at 20°C. 66% and 44% activity remained after incubation at 30°C and 40°C for 30 min. pH 7 and pH 9 incubation would obviously reduce the ATPase activity. In vivo functionality of gsiA was determined by constructing gene deletion strains...
2017: BioMed Research International
https://www.readbyqxmd.com/read/28684274/construction-of-high-level-prokaryotic-expression-and-purification-system-of-pd-l1-extracellular-domain-by-using-escherichia-coli-host-cell-machinery
#17
Muhammad Kalim, Jie Chen, Shenghao Wang, Caiyao Lin, Saif Ullah, Keying Liang, Qian Ding, Shuqing Chen, Jinbiao Zhan
Programmed cell death 1 ligand 1 (PD-L1) is a trans-membrane protein highly expressed on the membrane of cancer cell, which binds inhibitory receptor of PD-1 on the T cells and attenuates anti-tumor immune response.The strategy of blocking PD1 and PD-L1 interaction has been widely used for anti-cancer drug development. The DNA encoding extracellular domain of PD-L1wascloned and expressed with the pET30(+) and Escherichia coli BL21(DE3) system. Cloning of PD-L1 extracellular domain was confirmed by PCR and enzymatic digestion...
July 3, 2017: Immunology Letters
https://www.readbyqxmd.com/read/28682407/selective-protein-transport-through-ultra-thin-suspended-reduced-graphene-oxide-nanopores
#18
Dae-Sik Lee, Seokhan Park, Yong Duk Han, Jae Eun Lee, Hu Young Jeong, Hyun C Yoon, Moon Yeon Jung, Sang Ouk Kim, Sung-Yool Choi
The nanoporous free-standing graphene membrane is of great interest in high performance separation technology. In particular, the separation of biological molecules with similar sizes is one of the key challenges in the purification of biomaterials. Here, we report a reliable, cost-effective, and facile method for the fabrication of a graphene-based nanosieve and its application in the separation of similar-size proteins. A suspended reduced graphene oxide (rGO) nanosieve with ultra-thin, large-area, well-ordered, and dense 15 nm-sized pores was fabricated using block copolymer (BCP) lithography...
July 6, 2017: Nanoscale
https://www.readbyqxmd.com/read/28679762/aav-serotypes-have-distinctive-interactions-with-domains-of-the-cellular-receptor-aavr
#19
Sirika Pillay, Wei Zou, Fang Cheng, Andreas S Puschnik, Nancy L Meyer, Safder S Ganaie, Xuefeng Deng, Jonathan E Wosen, Omar Davulcu, Ziying Yan, John F Engelhardt, Kevin E Brown, Michael S Chapman, Jianming Qiu, Jan E Carette
Adeno-associated virus (AAV) entry is determined by its interactions with specific surface glycans and proteinaceous receptor(s). Adeno-associated virus receptor (AAVR; also named KIAA0319L) is an essential cellular receptor required for the transduction of vectors derived from multiple AAV serotypes including the evolutionary distant serotypes, AAV2 and AAV5. Here, we further biochemically characterize the AAV-AAVR interaction and define the domains within the ectodomain of AAVR that facilitate this interaction...
July 5, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28677644/2bc-non-structural-protein-of-enterovirus-a71-interacts-with-snare-proteins-to-trigger-autolysosome-formation
#20
Jeffrey K F Lai, I-Ching Sam, Pauline Verlhac, Joël Baguet, Eeva-Liisa Eskelinen, Mathias Faure, Yoke Fun Chan
Viruses have evolved unique strategies to evade or subvert autophagy machinery. Enterovirus A71 (EV-A71) induces autophagy during infection in vitro and in vivo. In this study, we report that EV-A71 triggers autolysosome formation during infection in human rhabdomyosarcoma (RD) cells to facilitate its replication. Blocking autophagosome-lysosome fusion with chloroquine inhibited virus RNA replication, resulting in lower viral titres, viral RNA copies and viral proteins. Overexpression of the non-structural protein 2BC of EV-A71 induced autolysosome formation...
July 4, 2017: Viruses
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