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https://www.readbyqxmd.com/read/28440667/camelus-dromedarius-glucose-transporter-4-in-silico-analysis-cloning-expression-purification-and-characterisation-in-e-coli
#1
Anwar Ahmed, Mohammed Arshad, Ajamaluddin Malik, Shama Parveen, Abdulrahman M Alsenaidy
Camels have exceptional carbohydrate metabolism as their plasma glucose level is high and have low whole body insulin sensitivity, similar to that observed in type 2 diabetes patients. We aimed at studing an important component of insulin signalling pathway, the GLUT4, in camel. Camelus dromedarius GLUT4 (CdGLUT4) CDS is 1530 nucleotide in length that encodes for a 55KDa protein. CdGLUT4 has 23 amino acid substitutions and 3N-glycosylation sites, compared to 2 in Human GLUT4. 3 D structures of CdGLUT4 and HsGLUT4 generated by homology modelling revealed conservation of characteristic signature motifs...
April 25, 2017: Archives of Physiology and Biochemistry
https://www.readbyqxmd.com/read/28439867/mapping-protein-protein-interactions-using-affinity-purification-and-mass-spectrometry
#2
Chin-Mei Lee, Christopher Adamchek, Ann Feke, Dmitri A Nusinow, Joshua M Gendron
The mapping of protein-protein interaction (PPI) networks and their dynamics are crucial steps to deciphering the function of a protein and its role in cellular pathways, making it critical to have comprehensive knowledge of a protein's interactome. Advances in affinity purification and mass spectrometry technology (AP-MS) have provided a powerful and unbiased method to capture higher-order protein complexes and decipher dynamic PPIs. However, the unbiased calling of nonspecific interactions and the ability to detect transient interactions remains challenging when using AP-MS, thereby hampering the detection of biologically meaningful complexes...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28439830/peptide-based-isolation-of-argonaute-protein-complexes-using-ago-app
#3
Judith Hauptmann, Gunter Meister
Argonaute (Ago) proteins bind small RNAs such as microRNAs (miRNAs) or short interfering RNAs (siRNAs), which guide them to distinct mRNAs for post-transcriptional gene silencing. Mammalian miRNA-guided gene silencing pathways mainly lead to translational repression and mRNA destabilization. To facilitate these processes, Ago proteins bind members of the GW protein family, which form central interaction platforms for the recruitment of downstream effector proteins. GW proteins use tryptophane residues (W) to bind to the surface of Ago proteins...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28438686/expression-purification-and-molecular-characterization-of-a-novel-endoglucanase-protein-from-bacillus-subtilis-sb13
#4
Xuefang Guan, Penglian Chen, Qingxian Xu, Lei Qian, Juqing Huang, Bin Lin
Bacillus subtilis strain SB13 which is isolated in our previous work was confirmed to produce endoglucanase. In this study, a novel endoglucanase gene (accession number: KX576676) was identified and cloned from SB13. Compared with other consensus sequence of reported endoglucanase genes in the GenBank database, this gene displays five differences (including T740C,A874G,A983G, T1210G and T1301C), which leading to five amino acid changes. Homology modeling has indicated that these five changes were located in the α-helix and random coil regions of the glycosyl hydrolase family 5 (GH5) domain, the random coil and β-sandwich of the type 3 carbohydrate-binding module (CBM3) domain, and the random coil domain...
April 21, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28438609/novel-replicons-and-trans-encapsidation-systems-for-hepatitis-c-virus-proteins-live-imaging-and-virus-host-interaction-proteomics
#5
Ovidiu Vlaicu, Tudor Selescu, Florin Pastrama, Cristian Munteanu, Laura Riva, Jean Dubuisson, Yves Rouille, Costin-Ioan Popescu
Proteomics and imaging techniques are used more and more in tandem to investigate the virus-host interaction. Herein we present novel replicons, methods and trans-encapsidation systems suitable for determination of Hepatitis C Virus (HCV) proteins interactomes and live imaging of viral proteins dynamics in HCV cell culture (HCVcc) system. To identify endogenous factors involved in the HCV life cycle, we constructed full-length functional replicons with affinity purification (AP) tags fused to NS2 and NS5A proteins...
April 21, 2017: Journal of Virological Methods
https://www.readbyqxmd.com/read/28438456/pirab-protein-from-xenorhabdus-nematophila-hb310-exhibits-a-binary-toxin-with-insecticidal-activity-and-cytotoxicity-in-galleria-mellonella
#6
Qing Yang, Jie Zhang, Tianhui Li, Shen Liu, Ping Song, Ziyan Nangong, Qinying Wang
PirAB (Photorhabdus insect-related proteins, PirAB) toxin was initially found in the Photorhabdus luminescens TT01 strain and has been shown to be a binary toxin with high insecticidal activity. Based on GenBank data, this gene was also found in the Xenorhabdus nematophila genome sequence. The predicted amino acid sequence of pirA and pirB in the genome of X. nematophila showed 51% and 50% identity with those gene sequences from P. luminescens. The purpose of this experiment is to identify the relevant information for this toxin gene in X...
April 21, 2017: Journal of Invertebrate Pathology
https://www.readbyqxmd.com/read/28437167/the-fgvps39-fgvam7-fgsso1-complex-mediates-vesicle-trafficking-and-is-important-for-the-development-and-virulence-of-fusarium-graminearum
#7
Bing Li, Luping Liu, Ying Li, Xin Dong, Haifeng Zhang, Huaigu Chen, Xiaobo Zheng, Zhengguang Zhang
Vesicle trafficking is an important event in eukaryotic organisms. Many proteins and lipids transported between different organelles or compartments are essential for survival. These processes are mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, Rab-GTPases, and multisubunit tethering complexes such as class C core vacuole or endosome tethering and homotypic fusion or vacuole protein sorting (HOPS). Our previous study has demonstrated that FgVam7, which encodes a SNARE protein involving in vesicle trafficking, plays crucial roles in growth, asexual or sexual development, deoxynivalenol production, and pathogenicity in Fusarium graminearum...
April 24, 2017: Molecular Plant-microbe Interactions: MPMI
https://www.readbyqxmd.com/read/28436004/engineering-of-an-h2-o2-auto-scavenging-in-vivo-cascade-for-pinoresinol-production
#8
Yongkun Lv, Xiaozhong Cheng, Guocheng Du, Jingwen Zhou, Jian Chen
Pinoresinol is a natural lignan with a high market value that has potential pharmacological and food supplement applications. Pinoresinol is currently isolated from plants, which suffers from low efficiency and yield. To produce pinoresinol from inexpensive and industrially available eugenol, an in vivo enzymatic cascade composed of vanillyl alcohol oxidase and peroxidase was designed, which scavenges H2 O2 automatically and eliminates protein purification and cofactor addition. Two peroxidases were screened and identified from Escherichia coli BL21 (DE3), and tested in the enzymatic cascade...
April 24, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28435877/design-and-function-of-biomimetic-multilayer-water-purification-membranes
#9
Shengjie Ling, Zhao Qin, Wenwen Huang, Sufeng Cao, David L Kaplan, Markus J Buehler
Multilayer architectures in water purification membranes enable increased water throughput, high filter efficiency, and high molecular loading capacity. However, the preparation of membranes with well-organized multilayer structures, starting from the nanoscale to maximize filtration efficiency, remains a challenge. We report a complete strategy to fully realize a novel biomaterial-based multilayer nanoporous membrane via the integration of computational simulation and experimental fabrication. Our comparative computational simulations, based on coarse-grained models of protein nanofibrils and mineral plates, reveal that the multilayer structure can only form with weak interactions between nanofibrils and mineral plates...
April 2017: Science Advances
https://www.readbyqxmd.com/read/28435537/integrated-platform-for-expedited-synthesis-purification-testing-of-small-molecule-libraries
#10
Aleksandra Baranczak, Noah P Tu, Jasmina Marjanovic, Philip A Searle, Anil Vasudevan, Stevan W Djuric
The productivity of medicinal chemistry programs can be significantly increased through the introduction of automation, leading to shortened discovery cycle times. Herein, we describe a platform that consolidates synthesis, purification, quantitation, dissolution, and testing of small molecule libraries. The system was validated through the synthesis and testing of two libraries of binders of polycomb protein EED, and excellent correlation of obtained data with results generated through conventional approaches was observed...
April 13, 2017: ACS Medicinal Chemistry Letters
https://www.readbyqxmd.com/read/28434999/smfret-experiments-of-the-rna-polymerase-ii-transcription-initiation-complex
#11
REVIEW
Nicole Malkusch, Thilo Dörfler, Julia Nagy, Tobias Eilert, Jens Michaelis
Single-molecule fluorescence and in particular single-molecule Förster Resonance Energy Transfer (smFRET(1)) is a powerful tool to provide real-time information on the dynamic architecture of large macromolecular structures such as eukaryotic transcription initiation complexes. In contrast to other structural biology methods, not only structural details, but dynamics transitions are revealed thus closing in on the underlying molecular mechanisms. Here, we describe a comprehensive quantitative biophysical toolbox which can be used for rigorous analysis of dynamic protein-nucleic acid complexes and is applied to the study of eukaryotic transcription initiation...
April 18, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28433193/regulation-of-the-catalytic-behavior-of-pullulanases-chelated-onto-nickel-ii-modified-magnetic-nanoparticles
#12
Jianfeng Wang, Zhongmei Liu, Zhemin Zhou
Chelating of pullulanases onto nickel (II)-modified magnetic nanoparticles results in one-step purification and immobilization of pullulanase, and facilitates the commercial application of pullulanase in industrial scale. To improve the catalytic behavior, especially the operational stability, of the nanocatalyst in consecutive batch reactions, we prepared various iminodiacetic acid-modified magnetic nanoparticles differed in surface polarity and spacer length, on which the His6-tagged pullulanases were chelated via nickel ions, and then studied the correlation between the MNPs surface property and the corresponding catalyst behavior...
June 2017: Enzyme and Microbial Technology
https://www.readbyqxmd.com/read/28433147/mucopolysaccharide-from-cuttlefish-purification-chemical-characterization-and-bioactive-potential
#13
Palaniappan Seedevi, Meivelu Moovendhan, Shanmugam Vairamani, Annaian Shanmugam
The sulfated mucopolysaccharide (GAG) was isolated from S. pharonis and the carbohydrate and protein content was found to be 62.4% and 3.9%. The disaccharide profile of sulfated GAG composed glucuronic acid, N-acetyl glucosamine and sulfate content by contributing 50.11%, 38.00% and 27.69% respectively. The carbon, hydrogen and nitrogen content of the sulfated GAG showed 14.80%, 1.68% and 2.99% respectively. The molecular weight of sulfated GAG was calculated as 27kDa and the structural characterization was done by Fourier Transform Infrared (FT-IR) and NMR Spectroscopy...
July 1, 2017: Carbohydrate Polymers
https://www.readbyqxmd.com/read/28432497/immobilization-of-ulp1-protease-on-nhs-activated-sepharose-a-useful-tool-for-cleavage-of-the-sumo-tag-of-recombinant-proteins
#14
Qiujin Liang, Zhengzhi Huang, Yuan Zhang, Hongtao Li
OBJECTIVE: To fabricate an active and stable enzyme through covalent immobilization, a Ubl-specific protease (Ulp1) was used to cleave small ubiquitin-like modifier (SUMO) fusion proteins. RESULTS: We immobilized Ulp1 on N-hydroxysuccinimide (NHS)-activated Sepharose with a coupling efficiency of 1.7 mg/ml. The immobilized Ulp1 maintains 95% substrate-cleavage ability and significantly enhances pH and thermal stability, especially can withstand pH of 10.5. Besides resistance against some small molecules, the immobilized Ulp1 can tolerate 15% (v/v) DMSO and 20% (v/v) ethanol...
April 21, 2017: Biotechnology Letters
https://www.readbyqxmd.com/read/28432439/characterization-of-a-functional-recombinant-human-creatine-kinase-mb-isoenzyme-prepared-by-tandem-affinity-purification-from-escherichia-coli
#15
Lihui Zou, Wen Su, Meng Wang, Wei Huang, Haijian Zhao, Enyi Zhang, Junhua Jin, Hongtao Xu, Fei Xiao
Creatine kinase isoform CK-MB has been widely applied as a biomarker of myocardial injury. While a variety of methods have been used to measure CK-MB activity or mass in clinical laboratories, a CK-MB standard is needed to eliminate between-method bias. Because the in vitro expression of human creatine kinase generates three isoenzymes, CK-MM, CK-MB, and CK-BB, it is important to establish an effective method to purify the isoform CK-MB from the mixture. In this study, we aimed at using tandem affinity purification (TAP) to purify recombinant CK-MB protein and evaluate its value in clinical laboratories...
April 21, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28432124/regulation-of-neurite-morphogenesis-by-interaction-between-r7-regulator-of-g-protein-signaling-complexes-and-g-protein-subunit-g%C3%AE-13
#16
Stephanie L Scherer, Matthew D Cain, Stanley M Kanai, Kevin M Kaltenbronn, Kendall J Blumer
The R7 regulator of G protein signaling family (R7-RGS) critically regulates nervous system development and function. Mice lacking all R7-RGS subtypes exhibit diverse neurological phenotypes, and humans bearing mutations in the retinal R7-RGS isoform RGS9-1 have vision deficits. Although each R7-RGS subtype forms heterotrimeric complexes with Gβ5 and R7-RGS binding protein (R7BP) that regulate G protein-coupled receptor signaling by accelerating deactivation of Gi/o α-subunits, several neurological phenotypes of R7-RGS knockout mice are not readily explained by dysregulated Gi/o signaling...
April 21, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28431999/novel-method-to-rapidly-and-efficiently-lyse-escherichia-coli-for-the-isolation-of-recombinant-protein
#17
Himanshu Joshi, Vikas Jain
Rapid and high-throughput protein purification methods are required to explore structure and function of several uncharacterized proteins. Isolation of recombinant protein expressed in Escherichia coli strain BL21 (DE3) depends largely on the efficient and speedy bacterial cell lysis, which is considered as the bottleneck during protein purification. Cells are usually lysed by either sonication or high pressure homogenization, both of which are slow, require special equipment, lead to heat generation, and may result in loss of protein's biological activity...
April 18, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28431943/expression-and-purification-of-p70%C3%AE-ct104-s6-k-a-72-kda-c-terminal-truncated-p70s6-kinase-gst-fusion-protein-in-bacterial-expression-system
#18
Younis Mohammad Hazari, Irfana Reshi, Mudasir Habib, Khalid Majid Fazili
The p70ΔCT104 S6K is a 421 amino acid residue long truncated form of p70S6 kinase, with 104 amino acids residues cleaved from the carboxyl terminal end of the original protein. The p70ΔCT104 S6K was cloned in E. coli DH5α and successfully expressed in E. coli BL21 (DE3) strain. Western blot with rabbit polyclonal anti-GST antibody was used to follow the protein during expression and purification. The protein purification was achieved by affinity chromatography using Glutathione resin-agarose beads, followed by chromatography on a spin concentration column...
April 18, 2017: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/28429419/semi-synthesis-of-murine-prion-protein-by-native-chemical-ligation-and-chemical-activation-for-preparation-of-polypeptide-%C3%AE-thioester
#19
Lei Shi, Huai Chen, Si-Yu Zhang, Ting-Ting Chu, Yu-Fen Zhao, Yong-Xiang Chen, Yan-Mei Li
Prions are suspected as pathogen of the fatal transmissible spongiform encephalopathies. Strategies to access homogenous prion protein (PrP) are required to fully comprehend the molecular mechanism of prion diseases. However, the polypeptide fragments from PrP show a high tendency to form aggregates, which is a gigantic obstacle of protein synthesis and purification. In this study, murine prion sequence 90 to 230 that is the core three-dimensional structure domain was constructed from three segments murine PrP (mPrP)(90-177), mPrP(178-212), and mPrP(213-230) by combining protein expression, chemical synthesis and chemical ligation...
April 21, 2017: Journal of Peptide Science: An Official Publication of the European Peptide Society
https://www.readbyqxmd.com/read/28429417/immobilization-methods-for-the-rapid-total-chemical-synthesis-of-proteins-on-microtiter-plates
#20
Robert Zitterbart, Michael Krumrey, Oliver Seitz
The chemical synthesis of proteins typically involves the solid-phase peptide synthesis of unprotected peptide fragments that are stitched together in solution by native chemical ligation (NCL). The process is slow, and throughput is limited because of the need for repeated high performance liquid chromatography purification steps after both solid-phase peptide synthesis and NCL. With an aim to provide faster access to functional proteins and to accelerate the functional analysis of synthetic proteins by parallelization, we developed a method for the high performance liquid chromatography-free synthesis of proteins on the surface of microtiter plates...
April 21, 2017: Journal of Peptide Science: An Official Publication of the European Peptide Society
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