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Purification his tagged protein

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https://www.readbyqxmd.com/read/29765994/heterologous-expression-purification-and-characterization-of-the-hspx-ppe44-and-esxv-proteins-of-mycobacterium-tuberculosis
#1
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/29736150/diagnostic-potential-of-brucella-melitensis-rev1-native-omp28-precursor-in-human-brucellosis
#2
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/29698455/isolation-of-mitochondria-from-saccharomyces-cerevisiae-using-magnetic-bead-affinity-purification
#3
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/29678627/structural-and-functional-insights-into-rha-p-a-bacterial-gh106-%C3%AE-l-rhamnosidase-from-novosphingobium-sp-pp1y
#4
Francesca Mensitieri, Federica De Lise, Andrea Strazzulli, Marco Moracci, Eugenio Notomista, Valeria Cafaro, Emiliano Bedini, Matthew Howard Sazinsky, Marco Trifuoggi, Alberto Di Donato, Viviana Izzo
α-L-Rhamnosidases (α-RHAs, EC 3.2.1.40) are glycosyl hydrolases (GHs) hydrolyzing terminal α-L-rhamnose residues from different substrates such as heteropolysaccharides, glycosylated proteins and natural flavonoids. Although the possibility to hydrolyze rhamnose from natural flavonoids has boosted the use of these enzymes in several biotechnological applications over the past decades, to date only few bacterial rhamnosidases have been fully characterized and only one crystal structure of a rhamnosidase of the GH106 family has been described...
April 17, 2018: Archives of Biochemistry and Biophysics
https://www.readbyqxmd.com/read/29626483/slyd-deficient-escherichia-coli-strains-a-highway-to-contaminant-free-protein-extraction
#5
Vladislav V Mokhonov, Ekaterina A Vasilenko, Ekaterina N Gorshkova, Irina V Astrakhantseva, Dmitry V Novikov, Viktor V Novikov
Binding of native bacterial protein SlyD to metal affinity matrices remains a major problem in affinity purification of His-tagged recombinant proteins from Escherichia coli cells. In this study, four novel E. coli strains that lack the expression of SlyD/SlyX, were engineered using λ-red mediated chromosomal deletion. The resultant mutant E. coli strains allow us to obtain SlyD-free proteins immediately after metal affinity chromatography, and eliminate additional purification processes. As a model protein, bispecific antibodies composed of anti-F4/80 VH H module and anti-TNF VH H module (MYSTI-2) were used...
April 4, 2018: Biochemical and Biophysical Research Communications
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
#6
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/29619665/expression-and-purification-of-the-main-component-contained-in-camel-milk-and-its-antimicrobial-activities-against-bacterial-plant-pathogens
#7
Abbas Tanhaeian, Farajollah Shahriari Ahmadi, Mohammad Hadi Sekhavati, Mojtaba Mamarabadi
Lactoferrin is the most dominant protein in milk after casein. This protein plays a crucial role in many biological processes including the regulation of iron metabolism, induction and modulation of the immune system, the primary defense against microorganisms, inhibiting lipid peroxidation and presenting antimicrobial activity against various pathogens such as parasites, fungi, bacteria, and viruses. The major antimicrobial effect of lactoferrin is related to its N-terminal tail where different peptides for instance lactoferricin and lactoferrampin which are important for their antimicrobial abilities are present...
April 4, 2018: Probiotics and Antimicrobial Proteins
https://www.readbyqxmd.com/read/29611635/molecular-cloning-expression-purification-and-osteoblasts-proliferation-activity-of-sika-deer-thymosin-beta10
#8
J Wang, M Liu, X Bai, H Zhang, Z Xu, D Zhao, Y Zhao
Thymosin beta 10 (Tβ10) is a member of the β-thymosin family. As an actin-binding peptide, thymosin β10 is involved in many important biological activities. Transcriptome sequencing results suggest that Tβ10 may play important roles in the growth of deer antler. In this study, Tβ10 cDNA was isolated from sika deer, and complete open reading frame consisting of 129 nucleotides was obtained by PCR amplification. The predicted peptide was 42 amino acids in length. The sdTβ10 cDNA was cloned and expressed in Escherichia coli resulting in a 6 kDa recombinant-His tagged protein...
December 2017: Polish Journal of Veterinary Sciences
https://www.readbyqxmd.com/read/29563556/near-native-site-specific-and-purification-free-protein-labeling-for-quantitative-protein-interaction-analysis-by-microscale-thermophoresis
#9
Tanja Bartoschik, Stefanie Galinec, Christian Kleusch, Katarzyna Walkiewicz, Dennis Breitsprecher, Sebastian Weigert, Yves A Muller, Changjiang You, Jacob Piehler, Thomas Vercruysse, Dirk Daelemans, Nuska Tschammer
MicroScale Thermophoresis (MST) is a frequently used method for the quantitative characterization of intermolecular interactions with several advantages over other technologies. One of these is its capability to determine equilibrium constants in solution including complex biological matrices such as cell lysates. MST requires one binding partner to be fluorescent, which is typically achieved by labeling target proteins with a suitable fluorophore. Here, we present a near-native, site-specific in situ labeling strategy for MST experiments that enables reliable measurements in cell lysates and that has distinct advantages over routine covalent labeling techniques...
March 21, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29551933/expression-of-aspergillus-niger-n5-5-in-e-coli-and-purification-and-identification-of-products
#10
Shuai Zhang, Yong Cao, Hao Cheng
Due to the feature of high hydrolysis, tannase is widely used in food, beverage, brewing and other fields. However, high cost in producing natural tannase makes it difficult to apply tannase to industry in a large-scale. Microbial expression systems can be used for preparing numerous amount of enzyme at low cost, so in this paper Aspergillus niger N5-5 was expressed using E. coli system. Specific primers were designed based on the Aspergillus niger N5-5 sequence N3 (GenBank, No.: KP677552), and tannase gene tan was promoted to carry 6 His tag and enzyme cutting site which contains NdeI/HindIII using PCR amplification...
December 2017: Saudi Journal of Biological Sciences
https://www.readbyqxmd.com/read/29545094/spherical-supported-membranes-as-platforms-for-screening-against-membrane-protein-targets
#11
V Vasilca, A Sadeghpour, S Rawson, L E Hawke, S A Baldwin, T Wilkinson, D Bannister, V L G Postis, M Rappolt, S P Muench, L J C Jeuken
Screening assays performed against membrane protein targets (e.g. phage display) are hampered by issues arising from protein expression and purification, protein stability in detergent solutions and epitope concealment by detergent micelles. Here, we have studied a fast and simple method to improve screening against membrane proteins: spherical-supported bilayer lipid membranes ("SSBLM"). SSBLMs can be quickly isolated via low-speed centrifugation and redispersed in liquid solutions while presenting the target protein in a native-like lipid environment...
May 15, 2018: Analytical Biochemistry
https://www.readbyqxmd.com/read/29509062/chitosan-cellulose-based-beads-for-the-affinity-purification-of-histidine-tagged-proteins
#12
Mingcong Shao, Lili Xiu, Haijiang Zhang, Jianying Huang, Xingwen Gong
Chitosan/cellulose-based beads (CCBs) for the affinity purification of histidine-tagged proteins were prepared from chitosan/cellulose dissolved in ionic liquid as a solvent, and their structures were characterized by Fourier transform infrared spectroscopy, transmission electron microscopy, and thermogravimetric analysis. The affinity purification was used to separate hexahistidine-tagged (his-tagged) enhanced green fluorescent protein (EGFP) from Escherichia coli. The results showed that Zn2+ -CCB exhibited more specific adsorption capacity toward the target protein compared with Ni2+ -CCB and Cu2+ -CCB...
April 21, 2018: Preparative Biochemistry & Biotechnology
https://www.readbyqxmd.com/read/29502205/heterologous-expression-purification-and-characterization-of-an-oligopeptidase-a-from-the-pathogen-leptospira-interrogans
#13
Prasannan V Anu, Madathiparambil G Madanan, Ananthakrishnan J Nair, Gangaprasad A Nair, Govinda Pillai M Nair, Perumana R Sudhakaran, Padikara K Satheeshkumar
Oligopeptidases are enzymes involved in the degradation of short peptides (generally less than 30 amino acids in size) which help pathogens evade the host defence mechanisms. Leptospira is a zoonotic pathogen and causes leptospirosis in mammals. Proteome analysis of Leptospira revealed the presence of oligopeptidase A (OpdA) among other membrane proteins. To study the role of oligopeptidase in leptospirosis, the OpdA of L. interrogans was cloned and expressed in Escherichia coli with a histidine tag (His-tag)...
April 2018: Molecular Biotechnology
https://www.readbyqxmd.com/read/29487134/the-n-terminal-domain-of-a-tick-evasin-is-critical-for-chemokine-binding-and-neutralization-and-confers-specific-binding-activity-to-other-evasins
#14
James R O Eaton, Yara Alenazi, Kamayani Singh, Graham Davies, Lucia Geis-Asteggiante, Benedikt Kessler, Carol V Robinson, Akane Kawamura, Shoumo Bhattacharya
Tick chemokine-binding proteins (evasins) are an emerging class of biologicals that target multiple chemokines and show anti-inflammatory activities in preclinical disease models. Using yeast surface display, we identified a CCL8-binding evasin, P672, from the tick Rhipicephalus pulchellus We found that P672 binds CCL8 and eight other CC-class chemokines with a Kd < 10 nm and four other CC chemokines with a Kd between 10 and 100 nm and neutralizes CCL3, CCL3L1, and CCL8 with an IC50 < 10 nm The CC chemokine-binding profile was distinct from that of evasin 1 (EVA1), which does not bind CCL8...
April 20, 2018: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29410083/transient-expression-of-a-bovine-leukemia-virus-envelope-glycoprotein-in-plants-by-a-recombinant-tbsv-vector
#15
Aiganym T Zhumabek, Laura S Abeuova, Nurzhan S Mukhametzhanov, Herman B Scholthof, Yerlan M Ramankulov, Shuga A Manabayeva
Plants offer a unique combination of advantages for the production of valuable recombinant proteins in a relatively short time. For instance, a variety of diagnostic tests have been developed that use recombinant antigens expressed in plants. The envelope glycoprotein gp51 encoded by Bovine leukemia virus (BLV) is one of the essential subunits for viral infectivity. It was indicated that the recombinant gp51 (rgp51) of BLV сan be used as an synthetic alternative antigen useful in the diagnosis of BLV infection in cattle...
May 2018: Journal of Virological Methods
https://www.readbyqxmd.com/read/29400346/unusual-stability-of-anabaena-sensory-rhodopsin-transducer-from-anabaena-pcc7120
#16
Vishwa D Trivedi, Tashmay S Jones, Renee P Walker
Advances in biotechnology generated wide range of microbial genome and their related protein database. Freshwater cyanobacterium Anabaena PCC7120 sensory rhodopsin, ASR in contrast to classical haloarchaeal sensory rhodopsins interacts with putative soluble transducer, ASRT. The 125 amino acid transducer exists as a soluble protein and is involved in photoreceptor binding. Recombinant DNA tools in biotechnology conventionally support the use of affinity tags for ease of protein purification and subsequent studies...
August 2017: The international journal of engineering and science
https://www.readbyqxmd.com/read/29360877/biotin-tagged-proteins-reagents-for-efficient-elisa-based-serodiagnosis-and-phage-display-based-affinity-selection
#17
Vaishali Verma, Charanpreet Kaur, Payal Grover, Amita Gupta, Vijay K Chaudhary
The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro...
2018: PloS One
https://www.readbyqxmd.com/read/29345069/automation-aided-optimization-of-cloning-expression-and-purification-of-enzymes-of-the-bacterial-sialic-acid-catabolic-and-sialylation-pathways-enzymes-for-structural-studies
#18
Sneha Bairy, Lakshmi Narayanan Gopalan, Thanuja Gangi Setty, Sathya Srinivasachari, Lavanyaa Manjunath, Jay Prakash Kumar, Sai R Guntupalli, Sucharita Bose, Vinod Nayak, Swagatha Ghosh, Nitish Sathyanarayanan, Rhawnie Caing-Carlsson, Weixiao Yuan Wahlgren, Rosmarie Friemann, S Ramaswamy, Muniasamy Neerathilingam
The process of obtaining a well-expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in vivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway...
January 17, 2018: Microbial Biotechnology
https://www.readbyqxmd.com/read/29340583/functional-analysis-of-the-helicobacter-pullorum-n-linked-protein-glycosylation-system
#19
Adrian J Jervis, Alison G Wood, Joel A Cain, Jonathan A Butler, Helen Frost, Elizabeth Lord, Rebecca Langdon, Stuart J Cordwell, Brendan W Wren, Dennis Linton
N-linked protein glycosylation systems operate in species from all three domains of life. The model bacterial N-linked glycosylation system from Campylobacter jejuni is encoded by pgl genes present at a single chromosomal locus. This gene cluster includes the pglB oligosaccharyltransferase responsible for transfer of glycan from lipid carrier to protein. Although all genomes from species of the Campylobacter genus contain a pgl locus, among the related Helicobacter genus only three evolutionarily related species (H...
April 1, 2018: Glycobiology
https://www.readbyqxmd.com/read/29318540/identification-of-proteolytic-cleavage-sites-of-epha2-by-membrane-type-1-matrix-metalloproteinase-on-the-surface-of-cancer-cells
#20
Keiji Kikuchi, Hiroko Kozuka-Hata, Masaaki Oyama, Motoharu Seiki, Naohiko Koshikawa
Proteolytic cleavage of membrane proteins can alter their functions depending on the cleavage sites. We recently demonstrated that membrane type 1 matrix metalloproteinase (MT1-MMP ) converts the tumor suppressor EphA2 into an oncogenic signal transducer through EphA2 cleavage. The cleaved EphA2 fragment that remains at the cell surface may be a better target for cancer therapy than intact EphA2. To analyze the cleavage site(s) of EphA2, we purified the fragments from tumor cells expressing MT1-MMP and Myc- and 6× His-tagged EphA2 by two-step affinity purification ...
2018: Methods in Molecular Biology
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