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Protein expression and purification

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https://www.readbyqxmd.com/read/29773106/-expression-and-purification-of-flt3-and-flt3-itd-mutated-proteins-in-hek293t-cells
#1
Fan Yang, Jiahui Du, Jie Wu, Puyu Yuan, Qian Su, Rong Han, Qiaocheng Qiu, Shengli Xue, Songbai Liu, Zhongwen Sun
Objective To construct the eukaryotic expression vectors of human fms related tyrosine kinase 3 (FLT3) gene and FLT3-internal tandem duplication (FLT3-ITD) mutants and purify the native proteins through immunoprecipitation from HEK293T cell lysates. Methods The cDNA fragments of FLT3wt and FLT3-ITD were amplified from bone marrow cells of healthy individuals and FLT3-ITD-mutated acute myeloid leukemia (AML) patients with specific primers, and the PCR products were cloned in CD530A-T2A-GFP expression vectors...
March 2018: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
https://www.readbyqxmd.com/read/29773100/-eukaryotic-expression-purification-of-human-igecepsilon2-4-protein-and-its-target-fishing
#2
Huihui Huang, Zheyan Wang, Zhenggen Yang, Zhen Li, Ping Wang, Wenqi Dong
Objective To construct eukaryotic expression vectors of human IgE heavy chain 2-4 region (IgECepsilon2-4) and purify the recombinant protein, and then capture its interacted proteins by surface plasmon resonance (SPR). Methods Three recombinant eukaryotic expression vectors of IgECepsilon2-4 containing different signal peptides were constructed and transiently transfected into HEK293FT suspension cells separately. The recombinant plasmid with the highest-level expression was selected to express the recombinant protein in a huge amount, and then the recombinant protein was purified by Ni-NTA affinity chromatography...
March 2018: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
https://www.readbyqxmd.com/read/29768182/enhanced-production-and-immunological-characterization-of-recombinant-west-nile-virus-envelope-domain-iii-protein
#3
Nagesh K Tripathi, Divyanshi Karothia, Ambuj Shrivastava, Swati Banger, Jyoti S Kumar
West Nile virus (WNV) is an emerging mosquito-borne virus which is responsible for severe and fatal encephalitis in humans and for which there is no licensed vaccine or therapeutic available to prevent infection. The envelope domain III protein (EDIII) of WNV was over-expressed in Escherichia coli and purified using a two-step chromatography process which included immobilized metal affinity chromatography and ion exchange chromatography. E. coli cells were grown in a bioreactor to high density using batch and fed-batch cultivation...
May 13, 2018: New Biotechnology
https://www.readbyqxmd.com/read/29768061/structural-re-positioning-in-silico-molecular-modelling-oxidative-degradation-and-biological-screening-of-linagliptin-as-adenosine-3-receptor-adora3-modulators-targeting-hepatocellular-carcinoma
#4
Bassam M Ayoub, Yasmeen M Attia, Mahmoud S Ahmed
Chemical entities with structural diversity were introduced as candidates targeting adenosine receptor with different clinical activities, containing 3,7-dihydro-1H-purine-2,6-dione, especially adenosine 3 receptors (ADORA3). Our initial approach started with pharmacophore screening of ADORA3 modulators; to choose linagliptin (LIN), approved anti-diabetic drug as Dipeptidyl peptidase-4 inhibitors, to be studied for its modulating effect towards ADORA3. This was followed by generation, purification, analytical method development, and structural elucidation of oxidative degraded product (DEG)...
December 2018: Journal of Enzyme Inhibition and Medicinal Chemistry
https://www.readbyqxmd.com/read/29765998/cloning-expression-purification-and-cd-analysis-of-recombinant-human-betatrophin
#5
Samaneh Gholami, Nematolah Gheibi, Reza Falak, Koorosh Goodarzvand Chegini
Background: Betatrophin is a member of the angiopoietin-like (ANGPTL) family that has been implicated in both triglyceride and glucose metabolism. The physiological functions and molecular targets of this protein remain largely unknown; hence, a purified available protein would aid study of the exact role of betatrophin in lipid or glucose metabolism. Methods: In this study, we cloned the full-length cDNA of betatrophin from a human liver cDNA library. Betatrophin was expressed in the pET-21b- E...
April 2018: Reports of Biochemistry & Molecular Biology
https://www.readbyqxmd.com/read/29765994/heterologous-expression-purification-and-characterization-of-the-hspx-ppe44-and-esxv-proteins-of-mycobacterium-tuberculosis
#6
Yousef Amini, Mohsen Tafaghodi, Saeid Amel Jamehdar, Zahra Meshkat, Bagher Moradi, Mojtaba Sankian
Background: Subunit vaccines are appropriate vaccine candidates for the prevention of some infections. In this study, three immunogenic proteins of Mycobacterium tuberculosis , including HspX, Ppe44, and EsxV as a new construction, were expressed alone and as a fusion protein to develop a new vaccine candidate against tuberculosis infection. Methods: To make the fusion protein, the three genes were linked together by AEAAAKEAAAKA linkers and inserted into pET21b and pET32b vectors...
April 2018: Reports of Biochemistry & Molecular Biology
https://www.readbyqxmd.com/read/29765812/enhanced-production-and-purification-of-recombinant-surface-array-protein-sap-for-use-in-detection-of-bacillus-anthracis
#7
Nidhi Puranik, N K Tripathi, V Pal, Ajay Kumar Goel
Surface array protein (Sap) can be an important biomarker for specific detection of Bacillus anthracis , which is released by the bacterium during its growth in culture broth. In the present work, we have cloned and expressed Sap in Escherichia coli . The culture conditions and cultivation media were optimized and used in batch fermentation process for scale up of Sap in soluble form. The recombinant Sap was purified employing affinity chromatography followed by diafiltration. The final yield of purified protein was 20 and 46 mg/l of culture during shake flasks and batch fermentation, respectively...
May 2018: 3 Biotech
https://www.readbyqxmd.com/read/29758321/production-of-soluble-bioactive-mouse-leukemia-inhibitory-factor-from-escherichia-coli-using-mbp-tag
#8
Yanan Guo, Miao Yu, Na Jing, Shoutao Zhang
Embryonic stem cells and induced pluripotent stem cells depend on one of cytokines called leukemia inhibitory factor (LIF) to retain their undifferentiated state and pluripotency. Nevertheless, further progresses of stem cell scientific investigation and its possible application are limited owing to the expense of commercial LIF. Here we introduced a simple, practical and high level expression of MBP-mouse LIF through Escherichia coli system which was bioactive. The mLIF cDNA was inserted into vector of pMAL-C2X in order to generate N-terminal MBP-mLIF recombinant proteins in the cytoplasm of Escherichia coli...
May 11, 2018: Protein Expression and Purification
https://www.readbyqxmd.com/read/29757589/the-effect-of-differentially-designed-fusion-proteins-to-elicit-efficient-anti-human-thyroid-stimulating-hormone-immune-responses
#9
Maysam Mard-Soltani, Mohamad Javad Rasaee, Saeed Khalili, Abdol-Karim Sheikhi, Mehdi Hedayati, Hossein Ghaderi-Zefrehi, Milad Alasvand
The production of human thyroid stimulating hormone (hTSH) immunoassays requires specific antibodies against hTSH which is a cumbersome process. Therefore, producing specific polyclonal antibodies against engineered recombinant fusion hTSH antigens would be of great significance. The best immunogenic region of the hTSH was selected based on in silico analyses and equipped with two different fusions. Standard methods were used for protein expression, purification, verification, structural evaluation, and immunizations of the white New Zealand rabbits...
April 2018: Iranian Journal of Allergy, Asthma, and Immunology
https://www.readbyqxmd.com/read/29752414/ligand-binding-and-activation-properties-of-the-purified-bacterial-cyclic-nucleotide-gated-channel-sthk
#10
Philipp A M Schmidpeter, Xiaolong Gao, Vikrant Uphadyay, Jan Rheinberger, Crina M Nimigean
Cyclic nucleotide-modulated ion channels play several essential physiological roles. They are involved in signal transduction in photoreceptors and olfactory sensory neurons as well as pacemaking activity in the heart and brain. Investigations of the molecular mechanism of their actions, including structural and electrophysiological characterization, are restricted by the availability of stable, purified protein obtained from accessible systems. Here, we establish that SthK, a cyclic nucleotide-gated (CNG) channel from Spirochaeta thermophila , is an excellent model for investigating the gating of eukaryotic CNG channels at the molecular level...
May 11, 2018: Journal of General Physiology
https://www.readbyqxmd.com/read/29751084/improved-high-yield-expression-purification-and-refolding-of-recombinant-mammalian-prion-proteins-under-aerosol-free-elevated-biological-safety-conditions
#11
Peter Rehbein, Harald Schwalbe
Production of recombinant prion proteins is of crucial relevance in food technology (analytical standards, assay development) but also in basic research, most importantly structural biology (NMR, X-ray diffraction). Structural approaches conveniently allow for sophisticated investigation of prion disease pathogenesis, but usually require large amounts of sample material. Recently, working with recombinant prion proteins has been recategorized to biosafety levels > S1 as infectious prions may readily be generated de novo and become airborne via aerosols...
May 8, 2018: Protein Expression and Purification
https://www.readbyqxmd.com/read/29747501/multi-proteomic-and-transcriptomic-analysis-of-oncogenic-%C3%AE-catenin-molecular-networks
#12
Rob M Ewing, Jing Song, Giridharan Gokulrangan, Sheldon Bai, Emily Bowler, Rachel Bolton, Paul Skipp, Yihua Wang, Zhenghe Wang
Dys-regulation of Wnt signalling is a frequent occurrence in many different cancers. Oncogenic mutations of CTNNB1/β-catenin, the key nuclear effector of canonical Wnt signalling, lead to accumulation and stabilization of β-catenin protein with diverse effects in cancer cells. Although the transcriptional response to Wnt/β-catenin signaling activation has been widely studied, an integrated understanding of the effects of oncogenic β-catenin on molecular networks is lacking. We used Affinity-Purification Mass-Spectrometry (AP-MS), label-free LC-MS/MS and RNA-Seq to compare protein-protein interactions, protein expression and gene-expression in colorectal cancer cells expressing mutant/oncogenic or wild-type β-catenin...
May 10, 2018: Journal of Proteome Research
https://www.readbyqxmd.com/read/29746246/conformationally-gated-electron-transfer-in-nitrogenase-isolation-purification-and-characterization-of-nitrogenase-from-gluconacetobacter-diazotrophicus
#13
Cedric P Owens, Faik A Tezcan
Nitrogenase is a complex, bacterial enzyme that catalyzes the ATP-dependent reduction of dinitrogen (N2 ) to ammonia (NH3 ). In its most prevalent form, it consists of two proteins, the catalytic molybdenum-iron protein (MoFeP) and its specific reductase, the iron protein (FeP). A defining feature of nitrogenase is that electron and proton transfer processes linked to substrate reduction are synchronized by conformational changes driven by ATP-dependent FeP-MoFeP interactions. Yet, despite extensive crystallographic, spectroscopic, and biochemical information on nitrogenase, the structural basis of the ATP-dependent synchronization mechanism is not understood in detail...
2018: Methods in Enzymology
https://www.readbyqxmd.com/read/29746241/fe-s-clusters-and-muty-base-excision-repair-glycosylases-purification-kinetics-and-dna-affinity-measurements
#14
Nicole N Nuñez, Chandrima Majumdar, Kori T Lay, Sheila S David
A growing number of iron-sulfur (Fe-S) cluster cofactors have been identified in DNA repair proteins. MutY and its homologs are base excision repair (BER) glycosylases that prevent mutations associated with the common oxidation product of guanine (G), 8-oxo-7,8-dihydroguanine (OG) by catalyzing adenine (A) base excision from inappropriately formed OG:A mispairs. The finding of an [4Fe-4S]2+ cluster cofactor in MutY, Endonuclease III, and structurally similar BER enzymes was surprising and initially thought to represent an example of a purely structural role for the cofactor...
2018: Methods in Enzymology
https://www.readbyqxmd.com/read/29744844/production-and-use-of-recombinant-a%C3%AE-for-aggregation-studies
#15
Tiernan T O'Malley, Sara Linse, Dominic M Walsh
The amyloid β-protein (Aβ) is believed to play a central role in Alzheimer's disease (AD) pathogenesis and there is great interest in understanding the process of Aβ aggregation, its underlying mechanism and the species generated during aggregation and their biological activity. Although Aβ has been studied for more than 30 years, analysis of its aggregation has been hampered by structural and chemical impurities. Here we provide a detailed protocol for the expression and purification of chemically and structurally homogeneous Aβ monomer...
2018: Methods in Molecular Biology
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
#16
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/29739070/high-level-prokaryotic-expression-and-purification-of-death-domain-superfamily-with-mbp-tag
#17
Kang Fang, Yajuan Li, Xueying Yin, Abdus Samad, Tengchuan Jin
BACKGROUND: Death domain superfamily are compact structural motifs that play critical roles in many biological processes including inflammation and apoptosis. Due to mediating protein-protein interactions, these death domains have a high tendency to form oligomers and are notoriously difficult to deal with when overexpressed in vitro. In this study, we found that maltose binding protein (MBP) is very effective in improving the behavior of many death domain superfamily members. METHODS: In order to achieve high-level expression of death domain superfamily in E...
April 1, 2018: Clinical Laboratory
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
#18
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/29738496/endogenous-purification-of-nr4a2-nurr1-identified-poly-adp-ribose-polymerase-1-as-a-prime-coregulator-in-human-adrenocortical-h295r-cells
#19
Erika Noro, Atsushi Yokoyama, Makoto Kobayashi, Hiroki Shimada, Susumu Suzuki, Mari Hosokawa, Tomohiro Takehara, Rehana Parvin, Hiroki Shima, Kazuhiko Igarashi, Akira Sugawara
Aldosterone is synthesized in zona glomerulosa of adrenal cortex in response to angiotensin II. This stimulation transcriptionally induces expression of a series of steroidogenic genes such as HSD3B and CYP11B2 via NR4A (nuclear receptor subfamily 4 group A) nuclear receptors and ATF (activating transcription factor) family transcription factors. Nurr1 belongs to the NR4A family and is regarded as an orphan nuclear receptor. The physiological significance of Nurr1 in aldosterone production in adrenal cortex has been well studied...
May 8, 2018: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/29736150/diagnostic-potential-of-brucella-melitensis-rev1-native-omp28-precursor-in-human-brucellosis
#20
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
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