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Protein Expression and Purification

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https://www.readbyqxmd.com/read/28728943/production-and-biophysical-characterization-of-a-mini-membrane-protein-ost4v23d-a-functionally-important-mutant-of-yeast-oligosaccharyltransferase-subunit-ost4p
#1
Bharat Chaudhary, Suman Mazumder, Smita Mohanty
N-linked glycosylation of proteins is an essential and highly conserved co- and post-translational protein modification reaction that occurs in all eukaryotes. Oligosaccharyltransferase (OST), a multi-subunit membrane-associated enzyme complex, carries out this reaction. In the central reaction, a carbohydrate group is transferred to the side chain of a consensus asparagine residue in the newly synthesized protein. Genetic defects in humans cause a series of disorders known as congenital disorders of glycosylation (CDG) that include mental retardation, developmental delay, hypoglycemia etc...
July 17, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28712957/secretory-expression-of-%C3%AE-mannanase-from-bacillus-circulans-nt-6-7-in-lactobacillus-plantarum
#2
Kwankanit Intaratrukul, Sunee Nitisinprasert, Thu-Ha Nguyen, Dietmar Haltrich, Suttipun Keawsompong
The β-mannanase gene of Bacillus circulans NT 6.7 was successfully cloned in Lactobacillus plantarum WCFS1 using the pSIP403 expression vector and secreted to the supernatant rather than accumulated in the cells. The highest activity was achieved by controlling the pH at 6 during cultivation. Maximum mannanase activities detected in the supernatant and cell-free extract of 200 ml MRS broth were 8.2 and 0.86 U/ml, respectively. Enzyme activity in the supernatant increased to 27 U/ml by fermentation in a 5-L bioreactor with automatic pH control...
July 13, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28712956/refolding-of-a-novel-cholesterol-oxidase-from-pimelobacter-simplex-reveals-dehydrogenation-activity
#3
Hui-Min Qin, Jian-Wen Wang, Qianqian Guo, Songtao Li, Panpan Xu, Zhangliang Zhu, Dengyue Sun, Fuping Lu
Cholesterol oxidases, which catalyze the degradation of cholesterol to cholest-4-en-3-one, are widely used in the pharmaceutical and food processing industries. The cholesterol oxidase from Pimelobacter simplex (PsChO3) was transformed into E. coli BL21(DE3), but it was expressed mainly as inclusion bodies, and any soluble PsChO3 failed to bind to Ni-NTA resin. To overcome this obstacle, we devised a simple yet efficient purification and refolding process using 8 M urea for the solubilization of PsChO3 and achieved a high yield of the enzyme in its active form...
July 13, 2017: Protein Expression and Purification
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
#4
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/28711731/insulin-chains-as-efficient-fusion-tags-for-prokaryotic-expression-of-short-peptides
#5
Ligang Deng, Xiaoying Xue, Cangjie Shen, Xiaohan Song, Chunyang Wang, Nan Wang
Insulin chains are usually expressed in Escherichia coli as fusion proteins with different tags, including various low molecular weight peptide tags. The objective of this study was to determine if insulin chains could facilitate the recombinant expression of other target proteins, with an emphasis on low molecular weight peptides. A series of short peptides were fused to mini-proinsulin, chain B or chain A, and induced for expression in Escherichia coli. All the tested peptides including glucagon-like peptide 1 (GLP-1), a C-terminal extended GLP-1, oxyntomodulin, enfuvirtide, linaclotide, and an unstructured artificial peptide were expressed with reasonable yields, identified by Tricine-SDS-PAGE and immunoblotting...
July 13, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28711732/high-level-expression-and-purification-of-a-mollusca-endoglucanase-from-ampullaria-crossean-in-pichia-pastoris
#6
Xiuping Su, Xinwei Geng, Meixian Fu, Yeqing Wu, Longfei Yin, Fukun Zhao, Wei Chen
EG27I is an endogenous glucanase belonging to glycoside hydrolase family (GHF) 45 from the Mollusca, Ampullaria crossean. In this study, the EG27I mature peptide gene fused to HFBII secretion signal of Trichoderma reesei was expressed under GAP promoter of Pichia pastoris in SMD1163 strain. Bioactive EG27I with a molecular weight of 27 kDa was successfully expressed and secreted into the culture medium. When high cell density fermentation of the recombinant Pichia pastoris was performed by a fed-batch strategy for totally 132 h in a 7...
July 12, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28711730/purification-and-characterization-of-a-long-acting-ciliary-neurotrophic-factor-via-genetically-fused-with-an-albumin-binding-domain
#7
Longfu Xu, Chun Zhang, Liping Liu, Yao Zhang, Qi Wang, Jian Wang, Yongdong Liu, Zhiguo Su
Ciliary neurotrophic factor (CNTF) is a promising candidate for the treatment of neurodegenerative or metabolic diseases, but suffers rapid clearance in body. Herein we constructed a new long-acting recombinant human CNTF (rhCNTF) by genetic fusion with an albumin-binding domain (ABD) through a flexible peptide linker, hoping to endow the new molecule prolonged serum circulation time by binding with endogenous human serum albumin (HSA) and then utilizing the naturally long-half-life property of HSA. This fused protein rhCNTF-ABD was expressed in Escherichia coli mainly in the soluble form and purified through a two-step chromatography, with purity of 95% and a high yield of 90-100 mg/L culture...
July 12, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28709863/dissociation-and-purification-of-the-endogenous-membrane-bound-vo-complex-from-pichia-pastoris
#8
Sumei Li, Tao Hong, Kun Wang, Yinghong Lu, Min Zhou
Most proteins occur and function in complexes rather than as isolated entities in membranes. In most cases macromolecules with multiple subunits are purified from endogenous sources. In this study, an endogenous membrane-protein complex was obtained from Pichia pastoris, which can be grown at high densities to significantly improve the membrane protein yield. We successfully isolated the membrane-bound Vo complex of V-ATPase from P. pastoris using a fusion FLAG tag attached to the C-terminus of subunit a to generate the vph-tag strain, which was used for dissociation and purification...
July 12, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28690182/functional-characterization-of-a-soluble-nadph-cytochrome-p450-reductase-from-fusarium-graminearum
#9
Thomas Etzerodt, Karl Wetterhorn, Giuseppe Dionisio, Ivan Rayment
Fusarium head blight is a devastating disease in wheat caused by some fungal pathogens of the Fusarium genus mainly F. graminearum, due to accumulation of toxic trichothecenes. Most of the trichothecene biosynthetic pathway has been mapped, although some proteins of the pathway remain uncharacterized, including an NADPH-cytochrome P450 reductase. We subcloned a F. graminearum cytochrome P450 reductase that might be involved in the trichothecene biosynthesis. It was expressed heterologously in E. coli as N-terminal truncated form with an octahistidine tag for purification...
July 6, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28673733/enhanced-soluble-production-of-cholera-toxin-b-subunit-in-escherichia-coli-by-co-expression-of-skp-chaperones
#10
Yuanpeng Zhang, Xuwen Qiao, Xiaoming Yu, Jin Chen, Liting Hou, Zhixiang Bi, Qisheng Zheng, Jibo Hou
The cholera toxin B subunit (CTB) is a nontoxic portion of the cholera toxin that retains mucosal adjuvant properties. Expression of CTB in Escherichia coli is difficult as CTB aggregates and accumulates as insoluble inclusion bodies. To remedy this problem, the periplasmic chaperone, SKP, was investigated as possible co-expression partner to increase the solubility of recombinant CTB (rCTB) in E. coli. The result showed co-expression of SKP enhanced the soluble expression of rCTB in E. coli. Moreover, soluble rCTB was successfully expressed and secreted into the periplasmic space through the direction of the LTB leader signal...
June 30, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28669870/secretory-expression-purification-and-functional-characterization-of-17%C3%AE-hydroxysteroid-dehydrogenase-type-1-from-mammalian-hek293t-cells
#11
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/28668497/a-fusion-protein-of-the-synthetic-igg-binding-domain-and-aequorin-expression-and-purification-from-e-%C3%A2-coli-cells-and-its-application
#12
Satoshi Inouye, Yuiko Sahara-Miura
Aequorin is a Ca(2+)-binding photoprotein that is a complex of apoaequorin (apoAQ) and 2-peroxycoelenterazine. In this study, the fusion protein (ZZ-apoAQ) composed of the synthetic IgG-binding domain (ZZ domain) derived from Staphylococcus aureus protein A and apoAQ was expressed into the periplasmic space of Escherichia coli cells. ZZ-apoAQ was highly purified using Ni-chelate affinity chromatography followed by IgG affinity chromatography. ZZ-AQ was prepared from purified ZZ-apoAQ by incubation with coelenterazine and was characterized, including its luminescence properties...
June 28, 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
#13
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/28651975/production-of-coagulation-factor-vii-in-human-cell-lines-sk-hep-1-and%C3%A2-hkb-11
#14
Marcela Cristina Corrêa de Freitas, Aline de Sousa Bomfim, Amanda Mizukami, Virgínia Picanço-Castro, Kamilla Swiech, Dimas Tadeu Covas
Recombinant factor VII (rFVII) is the main therapeutic choice for hemophilia patients who have developed inhibitory antibodies against conventional treatments (FVIII and FIX). Because of the post-translational modifications, rFVII needs to be produced in mammalian cell lines. In this study, for the first time, we have shown efficient rFVII production in HepG2, Sk-Hep-1, and HKB-11 cell lines. Experiments in static conditions for a period of 96 h showed that HepG2-FVII produced the highest amounts of rhFVII, with an average of 1843 ng/mL...
June 23, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28651974/variants-of-ppulcc-a-multi-dye-decolorizing-laccase-from-pleurotus-pulmonarius-expressed-in-pichia-pastoris
#15
Christoph J Behrens, Diana Linke, Aldrige B Allister, Katerina Zelena, Ralf G Berger
A laccase of the basidiomycete Pleurotus pulmonarius (PpuLcc) possessed strong decolorizing abilities towards artificial and natural dyes. The PpuLcc was purified from the culture supernatant via FPLC, and the corresponding gene cloned and expressed in Pichia pastoris GS115. To examine the impact of the C-terminal tail region and the signal peptide on the recombinant expression of PpuLcc, a non-modified version or different truncations (-2, -5, -13 AA) of the target protein were combined with different secretion signals...
June 23, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28648403/genetic-determinant-of-bacillus-pumilus-lipase-lethality-and-its-application-as-positive-selection-cloning-vector-in-escherichia-coli
#16
Nobalanda Betty Mabizela-Mokoena, Shonisani Wendy Limani, Ignatious Ncube, Lizelle Ann Piater, Derek Litthauer, Mulalo Bethuel Nthangeni
Positive selection vectors carry genes that upon expression produce proteins that cause host cell deaths. Insertion of foreign DNA fragments within the ORF of the gene disrupts the lethal effect of the expressed protein. This study described the cloning of Family I.4 Bacillus pumilus lipase gene whose expressed protein is toxic and lethal to Escherichia coli JM109 (DE3) cells. The determinant of toxicity was identified through Error-prone PCR to be the nature of amino acid residue resident at position 28 of the mature lipase protein...
June 23, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28642005/expression-purification-and-crystallization-of-type-1-isocitrate-dehydrogenase-from-trypanosoma-brucei-brucei
#17
Xinying Wang, Daniel Ken Inaoka, Tomoo Shiba, Emmanuel Oluwadare Balogun, Stefan Allmann, Yoh-Ichi Watanabe, Michael Boshart, Kiyoshi Kita, Shigeharu Harada
Isocitrate dehydrogenases (IDHs) are metabolic enzymes that catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate. Depending on the electron acceptor and subcellular localization, these enzymes are classified as NADP(+)-dependent IDH1 in the cytosol or peroxisomes, NADP(+)-dependent IDH2 and NAD(+)-dependent IDH3 in mitochondria. Trypanosoma brucei is a protozoan parasite that causes African sleeping sickness in humans and Nagana disease in animals. Here, for the first time, a putative glycosomal T...
June 20, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28625912/isolation-of-recombinant-human-untagged-glyceraldehyde-3-phosphate-dehydrogenase-from-e-%C3%A2-coli-producer-strain
#18
K V Barinova, M A Eldarov, E V Khomyakova, V I Muronetz, E V Schmalhausen
The goal of the present work was expression of human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) without additional tag constructions in E. coli cells and elaboration of the procedure for purification of untagged hGAPDH from the extract of the producer cells. We present a simple method for purification of untagged hGAPDH including ammonium sulfate fractionation and gel filtration on a G-100 Sephadex column. The method allows isolation of 2 mg of pure hGAPDH from 600 ml of cell culture (7 g of the cell biomass)...
June 15, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28625911/white-shrimp-litopenaeus-vannamei-recombinant-lactate-dehydrogenase-biochemical-and-kinetic-characterization
#19
Ambar A Fregoso-Peñuñuri, Elisa M Valenzuela-Soto, Ciria G Figueroa-Soto, Alma B Peregrino-Uriarte, Manuel Ochoa-Valdez, Lilia Leyva-Carrillo, Gloria Yepiz-Plascencia
Shrimp lactate dehydrogenase (LDH) is induced in response to environmental hypoxia. Two protein subunits deduced from different transcripts of the LDH gene from the shrimp Litopenaeus vannamei (LDHvan-1 and LDHvan-2) were identified. These subunits are expressed by alternative splicing. Since both subunits are expressed in most tissues, the purification of the enzyme from the shrimp will likely produce hetero LDH containing both subunits. Therefore, the aim of this study was to overexpress, purify and characterize only one subunit as a recombinant protein, the LDHvan-2...
June 15, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28624494/antibacterial-activity-and-phospholipid-recognition-of-the-recombinant-defensin-j1-1-from-capsicum-genus
#20
Francisco Guillén-Chable, Iván Arenas-Sosa, Ignacio Islas-Flores, Gerardo Corzo, Cynthia Martinez-Liu, Georgina Estrada
The gene of the four disulfide-bridged defensin J1-1 from Capsicum was cloned into the expression vector pQE30 containing a 6His-tag as fusion protein. This construct was transfected into Origami strain of Escherichia coli and expressed after induction with isopropyl thiogalactoside (IPTG). The level of expression was 4 mg/L of culture medium, and the His-tagged recombinant defensin (HisXarJ1-1) was expressed exclusively into inclusion bodies. After solubilization, HisXarJ1-1 was purified by affinity and hydrophobic interaction chromatography...
June 15, 2017: Protein Expression and Purification
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