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Protein Expression and Purification

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https://www.readbyqxmd.com/read/27888023/integrated-process-for-the-purification-and-immobilization-of-the-envelope-protein-domain-iii-of-dengue-virus-type-2-expressed-in-rachiplusia-nu-larvae-and-its-potential-application-in-a-diagnostic-assay
#1
María Emilia Smith, Alexandra Marisa Targovnik, Julieta Cerezo, María Alejandra Morales, María Victoria Miranda, Julián Rodríguez Talou
Dengue incidence has grown dramatically in the last years, with about 40% of the world population at risk of infection. Recently, a vaccine developed by Sanofi Pasteur has been registered, but only in a few countries. Moreover, specific antiviral drugs are not available. Thus, an efficient and accurate diagnosis is important for disease management. To develop a low-cost immunoassay for dengue diagnosis, in the present study we expressed the envelope protein domain III of dengue virus type 2 in Rachiplusia nu larvae by infection with a recombinant baculovirus...
November 22, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27867058/application-of-strep-tactin-xt-for-affinity-purification-of-twin-strep-tagged-cb2-a-g-protein-coupled-cannabinoid-receptor
#2
Alexei Yeliseev, Lioudmila Zoubak, Thomas G M Schmidt
Human cannabinoid receptor CB2 belongs to the class A of G protein-coupled receptor (GPCR). High resolution structural studies of CB2 require milligram quantities of purified, structurally intact protein. Here we describe an efficient protocol for purification of this protein using the Twin-Strep-tag/Strep-Tactin XT system. To improve the affinity of interaction of the recombinant CB2 with the resin, the double repeat of the Strep-tag was attached either to the N- or C-terminus of CB2 via a short linker. The CB2 was isolated at high purity from dilute solutions containing high concentrations of detergents, glycerol and salts, by capturing onto the Strep-Tactin XT resin, and was eluted from the resin under mild conditions upon addition of biotin...
November 17, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27864159/high-level-expression-and-efficient-refolding-of-therapeutically-important-recombinant-human-interleukin-3-hil-3-in-e-%C3%A2-coli
#3
Vikas Kumar Dagar, Adivitiya, Yogender Pal Khasa
Human interleukin-3 (hIL-3) is a pleiotropic cytokine that stimulates the differentiation and proliferation of multipotent hematopoietic cells thus making it a therapeutically important molecule. In this study, its poor expression yield was improved by addressing various upstream bottlenecks in E. coli heterologous system. The codon-optimized hIL-3 gene was cloned under various signal sequences and solubility enhancer fusion tags for its hyper-expression under a strong T7 promoter. The optimization of shake flask expression studies resulted in a hIL-3 protein concentration of 225 mg/L in the form of inclusion bodies (IBs)...
November 15, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27856402/the-expression-of-ntpdase1-and-2-of-leishmania-infantum-chagasi-in-bacterial-and-mammalian-cells-comparative-expression-refolding-and-nucleotidase-characterization
#4
M S Bastos, A Tremblay, J M Agripino, I L A Rabelo, L P Barreto, J Pelletier, J Lecka, A Silva-Júnior, G C Bressan, M R Almeida, J Sévigny, J L R Fietto
Visceral Leishmaniasis (VL) represents an important global health problem in several warm countries around the world. The main targets in this study are the two nucleoside triphosphate diphosphohydrolases (NTPDases) from Leishmania infantum chagasi that are the main etiologic agent of VL in the New World. These enzymes, called LicNTPDase1 and -2, are homologous to members 5 and 6 of the mammalian E-NTPDase/CD39 superfamily of enzymes. These enzymes hydrolyze nucleotides and accordingly can participate in the purine salvage pathways and in the modulation of purinergic signaling through the extracellular nucleotide-dependent host immune responses...
November 14, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27838376/expression-and-purification-of-biologically-active-recombinant-human-paraoxonase-1-from-a-drosophila-s2-stable-cell-line
#5
Hyeongseok Yun, Jiyeon Yu, Sumi Kim, Nari Lee, Jinhee Lee, Sungrae Lee, Nam Doo Kim, Chiho Yu, Jaerang Rho
Many pesticides and chemical warfare nerve agents are highly toxic organophosphorus compounds (OPs), which inhibit acetylcholinesterase activity. Human paraoxonase 1 (PON1) has demonstrated significant potential for use as a catalytic bioscavenger capable of hydrolyzing a broad range of OPs. However, there are several limitations to the use of human PON1 as a catalytic bioscavenger, including the relatively difficult purification of PON1 from human plasma and its dependence on the presence of hydrophobic binding partners to maintain stability...
November 9, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27826079/purified-stx-and-%C3%AE-phage-initiator-o-proteins-bind-specifically-to-two-different-origins-of-replication-in%C3%A2-vitro
#6
Katarzyna I Kozłowska, Joanna Tymecka-Mulik, Grzegorz Węgrzyn
The O protein is a crucial factor initiating the DNA replication of lambdoid bacteriophage. Efficient DNA replication of Shiga toxin-converting phage is necessary for effective production of Shiga toxin - main virulence factor of STEC strains. We developed an improved protocol for overproduction, bacterial cell lysis and purification of λO protein. With use of this method we have also isolated O proteins of Stx-phage P27 and 933W that were never purified before. Purified proteins were tested for their DNA binding activity and revealed a sequence specific interactions...
November 5, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27825980/expression-and-purification-of-native-and-functional-influenza-a-virus-matrix-2-proton-selective-ion-channel
#7
Elodie Desuzinges Mandon, Aurélien Traversier, Anne Champagne, Lorraine Benier, Stéphane Audebert, Sébastien Balme, Emmanuel Dejean, Manuel Rosa Calatrava, Anass Jawhari
Influenza A virus displays one of the highest infection rates of all human viruses and therefore represents a severe human health threat associated with an important economical challenge. Influenza matrix protein 2 (M2) is a membrane protein of the viral envelope that forms a proton selective ion channel. Here we report the expression and native isolation of full length active M2 without mutations or fusions. The ability of the influenza virus to efficiently infect MDCK cells was used to express native M2 protein...
November 5, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27815133/simple-purification-method-for-a-recombinantly-expressed-native-his-tag-free-aminopeptidase-a-from-lactobacillus-delbrueckii
#8
Timo Stressler, Coralie Tanzer, Jacob Ewert, Wolfgang Claaßen, Lutz Fischer
The aminopeptidase A (PepA; EC 3.4.11.7) is an intracellular exopeptidase present in lactic acid bacteria. The PepA cleaves glutamyl/aspartyl residues from the N-terminal end of peptides and can, therefore, be applied for the production of protein hydrolysates with an increased amount of these amino acids, which results in a savory taste (umami). The first PepA from a lactobacilli strain was recombinantly expressed in Escherichia coli in a recently published study and harbored a C-terminal His6-tag for easier purification...
November 1, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27789389/expression-purification-and-characterization-of-rop66-178-gtpase-from-arabidopsis-thaliana
#9
Yongheng Rong, Kun Wang, Renxing Shi, Xiaomin Hou, Chun-Hai Dong
The unique type of GTPases in plants, termed ROPs, are the small GTP-binding proteins involved in signal transduction which play important roles in regulation of hormonal response pathway, cell polarity, defense from plant pathogens, etc. In order to explore the regulation mechanism of AtROPs involved in, the purified ROPs were needed to explore the interactions of ROP GTPases with their regulators and effectors. In this study, the first ROP GTPase from Arabidopsis thaliana, AtROP66-178 was successfully expressed in Escherichia coli and obtained in high quality and purity through affinity chromatography and gel-filtration chromatography...
October 24, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27773761/expression-purification-immunogenicity-and-protective-efficacy-of-a-recombinant-nucleoside-hydrolase-from-leishmania-donovani-a-vaccine-candidate-for-preventing-cutaneous-leishmaniasis
#10
C Patrick McAtee, Christopher A Seid, Molly Hammond, Elissa Hudspeth, Brian P Keegan, Zhuyun Liu, Junfei Wei, Bin Zhan, Raul Arjona-Sabido, Vladimir Cruz-Chan, Eric Dumonteil, Peter J Hotez, Maria Elena Bottazzi
The nucleoside hydrolase gene from Leishmania donovani was cloned and expressed in Escherichia coli as a full length 36-kDa protein (LdNH36). Following lysis and extraction, the protein was purified by anion exchange and gel filtration chromatography. The purified protein had a molecular mass of approximately 36-kDa and was confirmed to be >99% pure. Using a nucleoside hydrolase assay, the protein was found to exhibit a Km of 741 ± 246 μM. Protein integrity was confirmed by lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS-PAGE), mass spectrometry (MS), and enzymatic assay...
October 20, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27756566/the-expression-and-purification-of-wssv134-from-white-spot-syndrome-virus-and-its-inhibitory-effect-on-caspase-activity-from-penaeus-monodon
#11
Thanunthon Bowornsakulwong, Walaiporn Charoensapsri, Triwit Rattanarojpong, Pongsak Khunrae
WSSV134 or VP36A protein of white spot syndrome virus was previously reported to be able to reduce apoptosis in Sf-9 cells transfected with caspase of Penaeus monodon (PmCasp). The protein was therefore believed to have a role in supporting the survival of WSSV inside the host cells during infection. However, the anti-apoptosis activity of WSSV134 involved in the inhibition of PmCasp is still unclear. In this study, we produced a recombinant WSSV134 (rWSSV134) and tested for its ability to inhibit PmCasp in vitro...
October 15, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27756565/purification-and-characterization-of-pseudomonas-aeruginosa-lasr-expressed-in-acyl-homoserine-lactone-free-escherichia-coli-cultures
#12
Andrés Corral Lugo, Abdelali Daddaoua, Alvaro Ortega, Bertrand Morel, Ana Isabel Díez Peña, Manuel Espinosa-Urgel, Tino Krell
Quorum sensing systems are essential for bacterial communication. We report here the purification and characterization of the Pseudomonas aeruginosa LasR quorum sensing regulator purified from lysates of E. coli cultures grown in the absence of added acyl-homoserine lactones (AHL). We show by isothermal titration calorimetry that LasR recognizes different AHLs with an affinity of approximately 1 μM. The affinity of LasR for its cognate 3-Oxo-C12-AHL was similar to that of other AHLs, indicating that this regulator has not evolved to preferentially recognize its cognate AHL...
October 15, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27751933/inducible-expression-of-trehalose-synthase-in-bacillus-licheniformis
#13
Youran Li, Zhenghua Gu, Liang Zhang, Zhongyang Ding, Guiyang Shi
Trehalose synthase (TreS) could transform maltose into trehalose via isomerization. It is a crucial enzyme in the process of trehalose enzymatical transformation. In this study, plasmid-based inducible expression systems were constructed to produce Thermomonospora curvata TreS in B. licheniformis. Xylose operons from B. subtilis, B. licheniformis and B. megaterium were introduced to regulate the expression of the gene encoding TreS. It was functionally expressed, and the BlsTs construct yielded the highest enzyme activity (12...
October 14, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27751932/effects-of-ionic-strength-on-inclusion-body-refolding-at-high-concentration
#14
Jakub Gabrielczyk, Jonas Kluitmann, Thorben Dammeyer, Hans-Joachim Jördening
For commercial applications refolding process must be fast, inexpensive and highly efficient. In the past many strategies for protein refolding were introduced. Still, simple refolding methods with high product concentrations are still rare. Refolding experiments were performed with fructosyltransferase (FTF, EC 2.4.1.162) from Bacillus subtilis NCIMB 11871 produced as inclusion bodies. Solubilizates were refolded with batch dialysis or by continuous exchange of dialysis buffers with variable ionic strength...
October 14, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27742254/recombinant-expression-purification-and-antimicrobial-activity-of-a-novel-antimicrobial-peptide-padef-in-pichia-pastoris
#15
De-Mei Meng, Jing-Fang Zhao, Xiao Ling, Hong-Xia Dai, Ya-Jun Guo, Xiao-Fang Gao, Bin Dong, Zi-Qi Zhang, Xin Meng, Zhen-Chuan Fan
The antimicrobial peptide PaDef was isolated from Mexican avocado fruit and was reported to inhibit the growth of Escherichia coli and Staphylococcus aureus in 2013. In this study, an N-terminal 6 × His tagged recombinant PaDef (rPaDef) with a molecular weight of 7.5 KDa, for the first time, was expressed as a secreted peptide in Pichia pastoris. The optimal culture condition for rPaDef expression was determined to be incubation with 1.5% methanol for 72 h at 28 °C under pH 6.0. Under this condition, the amount of the rPaDef accumulation reached as high as 79...
October 11, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27725246/cdna-isolation-and-functional-characterization-of-squalene-synthase-gene-from-ornithogalum-caudatum
#16
Ming Liu, Li-Na Li, Yi-Ting Pan, Jian-Qiang Kong
As the first step of ongoing efforts to investigate the genes responsible for the biosynthesis of steroidal saponins in the medicinal plant Ornithogalum caudatum, this investigation reported the cDNA isolation, prokaryotic expression and functional characterization of squalene synthase (SQS) gene from O. caudatum for the first time. Specifically, two unigenes showing high sequence identity to SQS were retrieved from RNA-Taq data, and then a full-length OcSQS1 corresponding to the two unigenes was isolated from O...
October 7, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27721079/a-non-cleavable-hexahistidine-affinity-tag-at-the-carboxyl-terminus-of-the-hiv-1-pr55-gag-polyprotein-alters-nucleic-acid-binding-properties
#17
Maria C Bewley, Lisa Reinhart, Matthew S Stake, Shorena Nadaraia-Hoke, Leslie J Parent, John M Flanagan
HIV Gag (Pr55(Gag)), a multidomain polyprotein that orchestrates the assembly and release of the human immunodeficiency virus (HIV), is an active target of antiretroviral inhibitor development. However, highly pure, stable, recombinant Pr55(Gag) has been difficult to produce in quantities sufficient for biophysical studies due to its susceptibility to proteolysis by cellular proteases during purification. Stability has been improved by using a construct that omits the p6 domain (Δp6). In vivo, p6 is crucial to the budding process and interacts with protein complexes in the ESCRT (Endosomal Sorting Complexes Required for Transport) pathway, it has been difficult to study its role in the context of Gag using in vitro approaches...
October 6, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27713060/affinity-purification-of-native-glycodelin-from-amniotic-fluid-for-biological-investigations-and-development-of-a-glycodelin-elisa-for-clinical-studies
#18
Steen Sørensen, Vibeke Myrhøj, Thanh Ha Nguyen, Per Aaslo, Young Bae Hansen
INTRODUCTION: Glycodelin is a glycoprotein with different oligosaccharides that are responsible for its diverse biological functions in contraception and immunosuppression. Therefore, it is necessary to have access to adequate amounts of glycodelin with retained carbohydrate structure for functional studies because the carbohydrate part can be lacking or be insufficient in recombinant glycodelin from prokaryotic and eukaryotic cell systems. METHODS AND RESULTS: Native glycodelin was purified from amniotic fluid by a series of affinity chromatography steps and had many glycosylated forms verified by mass spectrometry...
October 3, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27702601/towards-universal-approach-for-bacterial-production-of-three-finger-ly6-upar-proteins-case-study-of-cytotoxin-i-from-cobra-n-%C3%A2-oxiana
#19
M A Shulepko, E N Lyukmanova, Z O Shenkarev, P V Dubovskii, M V Astapova, A V Feofanov, A S Arseniev, Y N Utkin, M P Kirpichnikov, D A Dolgikh
Cytotoxins or cardiotoxins is a group of polycationic toxins from cobra venom belonging to the 'three-finger' protein superfamily (Ly6/uPAR family) which includes small β-structural proteins (60-90 residues) with high disulfide bond content (4-5 disulfides). Due to a high cytotoxic activity for cancer cells, cytotoxins are considered as potential anticancer agents. Development of the high-throughput production methods is required for the prospective applications of cytotoxins. Here, efficient approach for bacterial production of recombinant analogue of cytotoxin I from N...
October 1, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27693922/biological-function-analysis-of-monoclonal-antibodies-against-human-granulins-in%C3%A2-vitro-using-u251-cells-as-a-model
#20
Yanqing Li, Ya Li, Mingfu Ye, Dongyang Wang, Junli Zhao, Xiaohong Sun, Qinwen Mao, Haibin Xia
Progranulin (PGRN), a highly glycosylated, secreted 593 amino acid precursor protein, is a multifunctional molecule that is critical for early embryogenesis, wound repair, inflammatory and tumorigenesis. PGRN can be proteolytically cleaved into seven cysteine-rich granulin (Grn) peptides: G, F, B, A, C, D and E. Both PGRN and its constituent Grn peptides have been implicated in a wide variety of biological activities. However, their functions are far from clear, and the lack of granulin domain-specific antibodies has hindered the progress of the functional study of PGRN and Grns...
September 29, 2016: Protein Expression and Purification
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