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Affinity purification recombinant protein

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https://www.readbyqxmd.com/read/28735080/recombinant-truncated-e-protein-as-a-new-vaccine-candidate-against-nontypeable-h-influenzae-its-expression-and-immunogenic-evaluation
#1
Ava Behrouzi, Saeid Bouzari, Farzam Vaziri, Abolfazl Fateh, Parviz Afrough, Atefeh Davoudi Vijeh Motlagh, Seyed Davar Siadat
Protein E (PE) is a conserved entity observed in both nontypeable Haemophilus influenzae (NTHi) and encapsulated H. influenzae. This is a small surface lipoprotein, consisting of only 160 amino acids, involved in the adhesion of H. influenzae to various types of epithelial cells. A 384-bp-long fragment from NTHi PE was cloned into the prokaryotic expression vector pBAD-gIIIA. The recombinant protein was expressed with arabinose and then purified by affinity purification on an Ni-NTA agarose matrix. BALB/c mice were immunized by subcutaneous injection with purified recombinant truncated PE mixed with an alum adjuvant...
July 19, 2017: Microbial Pathogenesis
https://www.readbyqxmd.com/read/28731574/rapid-buffer-and-ligand-screening-for-affinity-chromatography-by-multiplexed-surface-plasmon-resonance-imaging
#2
Karin P M Geuijen, Daniëlle E J W van Wijk-Basten, David F Egging, Richard B M Schasfoort, Michel H Eppink
Protein purifications are often based on the principle of affinity chromatography, where the protein of interest selectively binds to an immobilized ligand. The development of affinity purification requires selecting proper wash and elution conditions. In recent years, miniaturization of the purification process is applied to speed up the development (e.g. microtiterplates, robocolumns). We have studied the application of surface plasmon resonance imaging (SPRi) as a tool to simultaneously screen many buffer conditions for wash and elution steps in an affinity-based purification process...
July 21, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28713777/a-novel-chimeric-endolysin-with-antibacterial-activity-against-methicillin-resistant-staphylococcus-aureus
#3
Hamed Haddad Kashani, Hossein Fahimi, Yasaman Dasteh Goli, Rezvan Moniri
Cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) and amidase are known as catalytic domains of the bacteriophage-derived endolysin LysK and were previously reported to show lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). In the current study, the in silico design and analysis of chimeric CHAP-amidase model was applied to enhance the stability and solubility of protein, which was achieved through improving the properties of primary, secondary and tertiary structures. The coding gene sequence of the chimeric CHAP-amidase was synthesized and subcloned into the pET-22(+) expression vector, and the recombinant protein was expressed in E...
2017: Frontiers in Cellular and Infection Microbiology
https://www.readbyqxmd.com/read/28713027/an-on-matrix-digestion-procedure-for-ap-ms-experiments-dissects-the-interplay-between-complex-conserved-and-serotype-specific-reactivities-in-dengue-virus-human-plasma-interactome
#4
Yassel Ramos, Vivian Huerta, Dayron Martín, Sucel Palomares, Alexis Yero, Dianne Pupo, Sebastien Gallien, Alejandro M Martín, Yasset Pérez-Riverol, Mónica Sarría, Osmany Guirola, Glay Chinea, Bruno Domon, Luis Javier González
The interactions between the four Dengue virus (DENV) serotypes and plasma proteins are crucial in the initial steps of viral infection to humans. Affinity purification combined with quantitative mass spectrometry analysis, has become one of the most powerful tools for the investigation on novel protein-protein interactions. Using this approach, we report here that a significant number of bait-interacting proteins do not dissociate under standard elution conditions, i.e. acid pH and chaotropic agents, and that this problem can be circumvented by using the "on-matrix" digestion procedure described here...
July 13, 2017: Journal of Proteomics
https://www.readbyqxmd.com/read/28711733/expression-and-purification-of-tat-ndrg2-recombinant-protein-and-evaluation-of-its-anti-proliferative-effect-on-lncap-cell-line
#5
Fahimeh Farokhinejad, Abbas Behzad Behbahani, Gholam Reza Rafiei Dehbidi, Mohammad Ali Takhshid
N-myc downstream regulated gene2 (NDRG2) belongs to tumor suppressor protein family of NDRG. Anti-proliferative and anti-metastasis of NDRG2 overexpression has been demonstrated in a number of tumors. The aim of this study was to fuse the gene of Trans Activator of Transcription (TAT) protein transduction domain with NDRG2 gene and express and purify TAT-NDRG2 fusion protein in order to investigate the effects of TAT-NDRG2 protein on proliferation and apoptosis of LNCaP prostate carcinoma cell line. pET28a-TAT-NDRG2 and pET28a-NDRG2 plasmids were constructed and transformed into E...
July 13, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28708446/elisa-reagent-coverage-evaluation-by-affinity-purification-tandem-mass-spectrometry
#6
Scott M Henry, Elissa Sutlief, Oscar Salas-Solano, John Valliere-Douglass
Host cell proteins (HCPs) must be adequately removed from recombinant therapeutics by downstream processing to ensure patient safety, product quality, and regulatory compliance. HCP process clearance is typically monitored by enzyme-linked immunosorbent assay (ELISA) using a polyclonal reagent. Recently, mass spectrometry (MS) has been utilized to identify specific HCP process impurities and monitor their clearance. Despite this capability, ELISA remains the preferred analytical approach due to its simplicity and throughput...
July 14, 2017: MAbs
https://www.readbyqxmd.com/read/28707664/-a-study-of-recombinant-human-sestrin-1-and-sestrin-2-proteins-produced-in-a-prokaryotic-system
#7
N Rai, R Kumar, Md A Haque, Md I Hassan, S Dey
Sestrins are highly conserved stress-inducible proteins capable of suppressing the production of ROS and signalling through mTORC1. Here we report a study of human sestrin1 (sesn1) and sestrin2 (sesn2) proteins produced in a pET28^(+) vector based prokaryotic system. Mass spectrometry analysis, western blot and surface plasmon resonance (SPR) of affinity purified sesn1 and sesn2 proteins confirmed their identity; biophysical characteristics were observed using circular dichroism (CD) showing that sesn1 and sesn2 have a predominant α-helical structure...
May 2017: Molekuliarnaia Biologiia
https://www.readbyqxmd.com/read/28706253/affinity-purification-of-erythropoietin-from-cell-culture-supernatant-combined-with-maldi-tof-ms-analysis-of-erythropoietin-n-glycosylation
#8
David Falck, Markus Haberger, Rosina Plomp, Michaela Hook, Patrick Bulau, Manfred Wuhrer, Dietmar Reusch
Erythropoietin (EPO) is a heavily glycosylated hormone whose recombinant forms are used for treatment of anaemia. EPO glycosylation is important for its pharmacological properties. An analytical workflow, which can determine EPO glycosylation in an accurate and high-throughput fashion from cell culture supernatant (CCS) in approximately 24 h, offers the possibility to follow changes during production. To address this challenge, we present a complete workflow consisting of protein purification, glycan release, sialic acid derivatization, solid phase extraction, matrix-assisted laser desorption/ionization - mass spectrometry (MALDI-MS) analysis and MassyTools data processing...
July 13, 2017: Scientific Reports
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
#9
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/28696676/alternative-affinity-ligands-for-immunoglobulins
#10
Nika Kruljec, Tomaž Bratkovič
The demand for recombinant therapeutic antibodies and Fc-fusion proteins is expected to increase in the years to come. Hence, extensive efforts are concentrated on improving the downstream processing. Especially the development of better affinity chromatography matrices, supporting robust, time- and cost-effective antibody purification, is warranted. With the advances in molecular design and high-throughput screening approaches from chemical and biological combinatorial libraries, novel affinity ligands representing alternatives to bacterial immunoglobulin (Ig)-binding proteins have entered the scene...
July 11, 2017: Bioconjugate Chemistry
https://www.readbyqxmd.com/read/28695523/purification-of-recombinant-human-parg-and-activity-assays
#11
Jean-Christophe Amé, Éléa Héberlé, Barbara Camuzeaux, Françoise Dantzer, Valérie Schreiber
The purification of Poly(ADP-ribose) glycohydrolase (PARG) from overexpressing bacteria Escherichia coli is described here to a fast and reproducible one chromatographic step protocol. After cell lysis, GST-PARG-fusion proteins from the crude extract are affinity purified by a Glutathione 4B Sepharose chromatographic step. The PARG proteins are then freed from their GST-fusion by overnight enzymatic cleavage using the preScission protease. As described in the protocol, more than 500 μg of highly active human PARG can be obtained from 1...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28695522/purification-of-recombinant-human-parp-3
#12
Jean-Christophe Amé, Barbara Camuzeaux, Françoise Dantzer, Valérie Schreiber
The purification of poly(ADP-ribose) polymerase-3 (PARP-3) from overexpressing cells (Sf9 insect cells, Escherichia coli) has been updated to a fast and reproducible two chromatographic steps protocol. After cell lysis, PARP-3 protein from the crude extract is affinity purified on a 3-aminobenzamide Sepharose™ chromatographic step. The last contaminants and the 3-methoxybenzamide used to elute PARP-3 from the previous affinity column are removed on the high-performance strong cations exchanger MonoQ™ matrix...
2017: Methods in Molecular Biology
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
#13
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/28670287/preparative-sds-page-as-an-alternative-to-his-tag-purification-of-recombinant-amelogenin
#14
Claire M Gabe, Steven J Brookes, Jennifer Kirkham
Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens, and in the development of therapeutics. Recombinant proteins are typically engineered with "tags" that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralization researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging...
2017: Frontiers in Physiology
https://www.readbyqxmd.com/read/28669870/secretory-expression-purification-and-functional-characterization-of-17%C3%AE-hydroxysteroid-dehydrogenase-type-1-from-mammalian-hek293t-cells
#15
Jiong Chen, Wei Feng, Yue Zhao
17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) mainly catalyzes the reduction of estrone into estradiol. The enzymatic conversion is a critical step in estradiol accumulation in breast tissue, which is a valuable prognosis index of breast cancer disease. However, the source of 17β-HSD1 for inhibitor design is limited. In this study, the fragment encoding human 17β-HSD1 was successfully cloned and expressed in human embryonic kidney (HEK) 293T mammalian cells. The recombinant protein was purified by immobilized metal ion affinity chromatography yielding above 17 mg of purified 17β-HSD1 protein per liter of cell culture, with a specific activity of 8...
June 29, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28668496/generation-of-nanobodies-against-slyd-and-development-of-tools-to-eliminate-this-bacterial-contaminant-from-recombinant-proteins
#16
Yaozhong Hu, Ema Romão, Didier Vertommen, Cécile Vincke, Francisco Morales-Yánez, Carlos Gutiérrez, Changxiao Liu, Serge Muyldermans
The gene for a protein domain, derived from a tumor marker, fused to His tag codons and under control of a T7 promotor was expressed in E. coli strain BL21 (DE3). The recombinant protein was purified from cell lysates through immobilized metal affinity chromatography and size-exclusion chromatography. A contaminating bacterial protein was consistently co-purified, even using stringent washing solutions containing 50 or 100 mM imidazole. Immunization of a dromedary with this contaminated protein preparation, and the subsequent generation and panning of the immune Nanobody library yielded several Nanobodies of which 2/3 were directed against the bacterial contaminant, reflecting the immunodominance of this protein to steer the dromedary immune response...
June 28, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28661426/rational-design-of-recombinant-papain-like-cysteine-protease-optimal-domain-structure-and-expression-conditions-for-wheat-derived-enzyme-triticain-%C3%AE
#17
Neonila V Gorokhovets, Vladimir A Makarov, Anastasiia I Petushkova, Olga S Prokopets, Mikhail A Rubtsov, Lyudmila V Savvateeva, Evgeni Yu Zernii, Andrey A Zamyatnin
Triticain-α is a papain-like cysteine protease from wheat (Triticumaestivum L.) that possesses activity towards toxic gluten-derived peptides, and was thus proposed as a novel therapeutic tool for celiac disease. We report an original approach employing rational design of domain architecture of Triticain-α and selection of the appropriate expression system for development of cheap and efficient protocol yielding active recombinant enzyme. The segregated catalytic domain of Triticain-α did not adopt native structure in bacteria, neither being expressed as a single protein nor upon conjugation or co-expression with extrinsic chaperones...
June 29, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/28658417/evaluation-of-recombinant-cryptosporidium-hominis-gp60-protein-and-anti-gp60-chicken-polyclonal-igy-for-research-and-diagnostic-purposes
#18
Valéria Chamas Miura, Sérgio Moraes Aoki, Paulo Peitl, Lilian Campos Pires, Priscila Dalmagro, Alex Akira Nakamura, Marcelo Vasconcelos Meireles
In this study, a method for expressing Cryptosporidium hominis GP60 glycoprotein in Escherichia coli for production of polyclonal anti-GP60 IgY in chickens was developed aiming future studies concerning the diagnosis, prevention and treatment of cryptosporidiosis. The full-length nucleotide sequence of the C. hominis gp60 gene was codon-optimized for expression in E. coli and was synthesized in pET28-a vector. Subcloning was performed on several different strains of BL21 E. coli. Temperature, time and inducer IPTG concentration assays were also performed and analyzed using SDS-PAGE...
June 22, 2017: Revista Brasileira de Parasitologia Veterinária, Brazilian Journal of Veterinary Parasitology
https://www.readbyqxmd.com/read/28645932/a-functional-nav1-7-navab-chimera-with-a-reconstituted-high-affinity-protx-ii-binding-site
#19
Ramkumar Rajamani, Sophie Wu, Iyoncy Rodrigo, Mian Gao, Simon Low, Lisa Megson, David Wensel, Rick Pieschl, Debra Post-Munson, John Watson, David R Langley, Michael Ahlijanian, Linda Bristow, James Herrington
NaV1.7 is genetically implicated in human pain perception. Rare gain of function mutations in NaV1.7 lead to spontaneous pain in humans whereas loss of function mutations result in congenital insensitivity to pain (CIP). Hence, agents that specifically modulate the function of NaV1.7 have the potential to yield novel therapeutics to treat pain. The complexity of the channel and the challenges to generate recombinant cell lines with high NaV1.7 expression have led to a templated target strategy approach employing chimeras with the bacterial channel, NavAb...
June 23, 2017: Molecular Pharmacology
https://www.readbyqxmd.com/read/28642366/saccharomyces-cerevisiae-red1-protein-exhibits-nonhomologous-dna-end-joining-activity-and-potentiates-hop1-promoted-pairing-of-double-stranded-dna
#20
Rucha Kshirsagar, Indrajeet Ghodke, Kalappa Muniyappa
Elucidation of the function of synaptonemal complex (SC) in <Saccharomyces cerevisiae> has mainly focused on in vivo analysis of recombination-defective meiotic mutants. Consequently, significant gaps remain in the mechanistic understanding of the activities of different SC proteins and the functional relationships among them. S. cerevisiae Hop1 and Red1 are essential structural components of the SC axial/lateral elements. Previous studies have demonstrated that Hop1 is a structure-specific DNA-binding protein that exhibits high affinity for the Holliday junction, promotes DNA bridging, condensation and pairing between duplex DNA molecules...
June 22, 2017: Journal of Biological Chemistry
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