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Protein Expression and Purification

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https://www.readbyqxmd.com/read/28214589/enhanced-expression-and-purification-of-camelid-single-domain-vhh-antibodies-from-classical-inclusion-bodies
#1
Maristella Maggi, Claudia Scotti
Single domain antibodies (sdAbs) are small antigen-binding domains derived from naturally occurring, heavy chain-only immunoglobulins isolated from camelid and sharks. They maintain the same binding capability of full-length IgGs but with improved thermal stability and permeability, which justifies their scientific, medical and industrial interest. Several described recombinant forms of sdAbs have been produced in different hosts and with different strategies. Here we present an optimized method for a time-saving, high yield production and extraction of a poly-histidine-tagged sdAb from Escherichia coli classical inclusion bodies...
February 15, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28192199/viral-hemorrhagic-septicemia-virus-glycoprotein-production-in-tobacco
#2
Nguyen-Quang-Duc Tien, Tae-Jung Kim, Tae-Geum Kim
Viral hemorrhagic septicemia virus (VHSV) causes mortality in numerous marine and freshwater fish species resulting in heavy losses in fish farming. The glycoprotein gene of VHSV was fused with the cholera toxin B subunit (CTB) and expressed transiently in leaf tissues of Nicotiana benthamiana via the agroinfiltration method. The glycoprotein gene was divided into two parts to improve assembly of CTB fusion proteins (CTB-VHSV99-235 and CTB-VHSV258-417). Production of CTB fusion proteins was confirmed in the agroinfiltrated leaf tissue by western blot analysis...
February 9, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28192198/molecular-cloning-purification-and-characterization-of-brugia-malayi-phosphoglycerate-kinase
#3
Ranjeet Kumar, Pawan Kumar Doharey, Jitendra Kumar Saxena, Sushma Rathaur
Phosphoglycerate kinase (PGK) is a glycolytic enzyme present in many parasites. It has been reported as a candidate molecule for drug and vaccine developments. In the present study, a full-length cDNA encoding the Brugia malayi 3-phosphoglycerate kinase (BmPGK) with an open reading frame of 1.3 kb was isolated and PCR amplified and cloned. The exact size of the BmPGK's ORF is 1377 bps. The BmPGK gene was subcloned into pET-28a (+) expression vector, the expressed enzyme was purified by affinity column and characterized...
February 9, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28189633/an-effective-and-simplified-do-stat-control-strategy-for-production-of-rabies-glycoprotein-in-pichia-pastoris
#4
L D Picotto, G H Sguazza, M A Tizzano, C M Galosi, S F Cavalitto, M R Pecoraro
The glycoprotein (G-protein) of rabies virus is responsible for viral attachment to the host cell surface and induces virus neutralization antibodies. In the present study, the G-protein gene of rabies virus CVS strain was cloned, sequenced and expressed in the yeast, Pichia pastoris, as a secreted protein, using a simplified DO-stat control feeding strategy. This strategy involves the addition of methanol when the dissolved oxygen (DO) level rises above the setpoint avoiding methanol accumulation and oxygen limitation...
February 9, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28188826/convenient-expression-purification-and-quantitative-liquid-chromatography-tandem-mass-spectrometry-based-analysis-of-tet2-5-methylcytosine-demethylase
#5
Mohit Jaiswal, Subhradeep Bhar, Harika Vemula, Swami Prakash, V K Chaithanya Ponnaluri, William G Gutheil, Mridul Mukherji
5-Methylcytosine within CpG islands in DNA plays a crucial role in epigenetic transcriptional regulation during metazoan development. Recently, it has been established that the Ten-Eleven Translocation (TET) family, Fe(II)- and 2-oxoglutarate (2OG/αKG)-dependent oxygenases initiate 5-methylcytosine demethylation by iterative oxidation reactions. Mutations in the TET2 gene are frequently detected in patients with myeloid malignancies. Here, we describe the cloning of untagged human TET2 demethylase using Gateway technology and its efficient expression in E...
February 8, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28185908/purification-and-biophysical-characterization-of-the-aimp2-dx2-protein
#6
Roshan Jha, Hye Young Cho, Ameeq Ul Mushtaq, Kiho Lee, Dae Gyu Kim, Sunghoon Kim, Young Ho Jeon
Besides their primary role in protein synthesis, aminoacyl-tRNA synthetases (AARSs) are involved in several non-canonical processes such as apoptosis, inflammation and angiogenesis through their interactions with various cellular proteins. Nine of these AARSs interact with three aminoacyl-tRNA synthetase interacting multifunctional proteins (AIMPs), forming a multi-synthetase complex (MSC) in eukaryotes. Among the three AIMPs, AIMP2 is involved in controlling cell proliferation and apoptosis. However, a splicing variant of AIMP2 lacking exon 2, referred to as AIMP2-DX2, is oncogenic and compromises the pro-apoptotic activity of AIMP2 by competing with it for p53 and TRAF2...
February 7, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28167250/purification-and-characterization-of-human-dehydrodolychil-diphosphate-synthase-dhdds-overexpressed-in-e-%C3%A2-coli
#7
Moshe Giladi, Ilan Edri, Michal Goldenberg, Hadas Newman, Roi Strulovich, Daniel Khananshvili, Yoni Haitin, Anat Loewenstein
Protein asparagine (N)-linked glycosylation is a post-translational modification that occurs in the endoplasmic reticulum; it plays an important role in protein folding, oligomerization, quality control, sorting, and transport. Accordingly, disorders of glycosylation may affect practically every organ system. Dehydrodolichyl diphosphate synthase (DHDDS) is an eukaryotic cis prenyltransferase (cis-PT) that catalyzes chain elongation of farnesyl diphosphate via multiple condensations with isopentenyl diphosphate to form dehydrodolichyl diphosphate, a precursor for the glycosyl carrier dolichylpyrophophate involved in N-linked glycosylation...
February 3, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28163214/preparative-refolding-of-small-monomeric-outer-membrane-proteins
#8
Tom Sebastian Schwarzer, Maria Hermann, Swati Krishnan, Friedrich C Simmel, Kathrin Castiglione
The outer membrane of gram-negative bacteria constitutes an important hurdle for the transport of hydrophobic molecules into the cell. Mass flux is often facilitated by various outer membrane proteins. These proteins are of biotechnological importance because they could help to improve the performance of whole-cell biocatalysts or be incorporated into artificial cell-like systems. The characterization and understanding of their transport properties greatly benefits from the possibility to express and purify these proteins...
February 2, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28161546/evaluation-of-changes-in-promoters-use-of-ucoes-and-chain-order-to-improve-the-antibody-production-in-cho-cells
#9
Maria Del Refugio Rocha-Pizaña, Guadalupe Ascencio-Favela, Brenda Maribell Soto-García, Margarita de la Luz Martinez-Fierro, Mario Moisés Alvarez
Therapy with biopharmaceuticals, mainly recombinant antibodies, offers patients higher life expectancy and better life quality than pharmacologic therapy. Countries with the highest scientific development are investing in this kind of therapy, and this is why the optimization of the production of these recombinant proteins would lead to their higher production and lower costs of the final product. Modifications in the use of promoters, the use of recombination regions, and the change in the order of the chains, are some of the genetic engineering changes that can increase the production of recombinant antibodies...
February 2, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28161544/purification-and-characterization-of-peptides-from-capsicum-annuum-fruits-which-are-%C3%AE-amylase-inhibitors-and-exhibit-high-antimicrobial-activity-against-fungi-of-agronomic-importance
#10
Layrana de Azevedo Dos Santos, Gabriel Bonan Taveira, Suzanna de Fátima Ferreira Ribeiro, Lídia da Silva Pereira, André de Oliveira Carvalho, Rosana Rodrigues, Antônia Elenir Amâncio Oliveira, Olga Lima Tavares Machado, Jucélia da Silva Araújo, Ilka Maria Vasconcelos, Valdirene Moreira Gomes
Proteins extracted from Capsicum annuum L. fruits were initially subjected to reversed-phase chromatography on HPLC, resulting in eight peptide-rich fractions. All the fractions obtained were tested for their ability to inhibit porcine trypsin and amylase from both human saliva and from larval insect in vitro. All fractions were also tested for their ability to inhibit growth of the phytopathogenic fungi. Several fractions inhibited the activity of human salivary amylase and larval insect amylase, especially fraction Fa5...
February 2, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28161545/%C3%AE-glucosidase-from-streptomyces-griseus-nanoparticle-immobilisation-and-alkyl-glucoside-synthesis
#11
P Kumar, B Ryan, G T M Henehan
A novel β-glucosidase from Streptomyces griseus was cloned and overexpressed in E. coli. The purified β-glucosidase (44 kDa) had a Km of 8.6 ± 0.5 mM and a Vmax of 217 ± 5.0 μmoles(-1)min(-1)mg at 37 °C, pH 7.2 with p-nitrophenyl-β-D glucopyranoside as substrate. The enzyme was characterised in terms of pH optimum (pH 6.9), temperature optimum (69 °C) and the influence of solvents and effectors. Purified S. griseus β-glucosidase was successfully immobilised, by simple absorption, onto zinc oxide (ZnO) nanoparticles without covalent modification...
February 1, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28153773/human-gpn1-purified-from-bacteria-binds-guanine-nucleotides-and-hydrolyzes-gtp-as-a-protein-dimer-stabilized-by-its-c-terminal-tail
#12
Rogelio González-González, José A Guerra-Moreno, Gema R Cristóbal-Mondragón, Violeta Romero, Sonia G Peña-Gómez, Gabriela M Montero-Morán, Samuel Lara-González, Andrés Hernández-Arana, Daniel A Fernández-Velasco, Mónica R Calera, Roberto Sánchez-Olea
The essential GTPase Gpn1 mediates RNA polymerase II nuclear targeting and controls microtubule dynamics in yeast and human cells by molecular mechanisms still under investigation. Here, we purified human HisGpn1 expressed as a recombinant protein in bacteria E. coli BL-21 (DE3). Affinity purified HisGpn1 eluted from a size exclusion column as a protein dimer, a state conserved after removing the hexa-histidine tail and confirmed by separating HisGpn1 in native gels, and in dynamic light scattering experiments...
January 30, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28137655/a-fully-automated-procedure-for-the-parallel-multidimensional-purification-and-nucleotide-loading-of-the-human-gtpases-kras-rac1-and-ralb
#13
Christopher H Gray, Jennifer Konczal, Mokdad Mezna, Shehab Ismail, Justin Bower, Martin Drysdale
Small GTPases regulate many key cellular processes and their role in human disease validates many proteins in this class as desirable targets for therapeutic intervention. Reliable recombinant production of GTPases, often in the active GTP loaded state, is a prerequisite for the prosecution of drug discovery efforts. The preparation of these active forms can be complex and often constricts the supply to the reagent intensive techniques used in structure base drug discovery. We have established a fully automated, multidimensional protein purification strategy for the parallel production of the catalytic G-domains of KRas, Rac1 and RalB GTPases in the active form...
January 28, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28109855/heterologous-expression-and-characterization-of-plant-taxadiene-5%C3%AE-hydroxylase-cyp725a4-in-escherichia-coli
#14
John Edward Rouck, Bradley Walters Biggs, Amogh Kambalyal, William R Arnold, Marjan De Mey, Parayil Kumaran Ajikumar, Aditi Das
Taxadiene-5α-Hydroxylase (CYP725A4) is a membrane-bound plant cytochrome P450 that catalyzes the oxidation of taxadiene to taxadiene-5α-ol. This oxidation is a key step in the production of the valuable cancer therapeutic and natural plant product, taxol. In this work, we report the bacterial expression and purification of six different constructs of CYP725A4. All six of these constructs are N-terminally modified and three of them are fused to cytochrome P450 reductase to form a chimera construct. The construct with the highest yield of CYP725A4 protein was then selected for substrate binding and kinetic analysis...
January 18, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28108349/expression-of-a-soluble-truncated-vargula-luciferase-in-escherichia-coli
#15
Eric A Hunt, Angeliki Moutsiopoulou, David Broyles, Trajen Head, Emre Dikici, Sylvia Daunert, Sapna K Deo
Marine luciferases are regularly employed as useful reporter molecules across a range of various applications. However, attempts to transition expression from their native eukaryotic environment into a more economical prokaryotic, i.e. bacterial, expression system often presents several challenges. Specifically, bacterial protein expression inherently lacks chaperone proteins to aid in the folding process, while Escherichia coli presents a reducing cytoplasmic environment in. These conditions contribute to the inhibition of proper folding of cysteine-rich proteins, leading to incorrect tertiary structure and ultimately inactive and potentially insoluble protein...
January 18, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28087367/production-of-recombinant-proteins-in-escherichia-coli-tagged-with-the-fusion-protein-cusf3h
#16
Teresa Vargas-Cortez, Jose Ruben Morones-Ramirez, Isaias Balderas-Renteria, Xristo Zarate
Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E...
January 11, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28089882/a-highly-efficient-modified-human-serum-albumin-signal-peptide-to-secrete-proteins-in-cells-derived-from-different-mammalian-species
#17
Carolina Attallah, Marina Etcheverrigaray, Ricardo Kratje, Marcos Oggero
Signal peptides (SPs) are key elements in the production of recombinant proteins; however, little information is available concerning different SP in mammalian cells other than CHO. In order to study the efficiency of different SPs to direct the traffic along the secretory pathway of the green fluorescence protein (GFP) and a scFv-Fc fusion protein; CHO-K1, HEK293 and NS0 cell lines were transfected in a transient and stable way. SP of human azurocidin (AZ), modified human albumin (mSA), modified Cricetulus griseus Ig kappa chain V III region MOPC 63 like (mIgκ C) and modified human Ig kappa chain V III region VG (mIgκ H) were evaluated...
January 10, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28089881/two-step-chromatographic-purification-of-glutathione-s-transferase-tagged-human-papillomavirus-type-16-e6-protein-and-its-application-for-serology
#18
Mei Ling Xu, Seung Cheol Kim, Hyoung Jin Kim, Woong Ju, Yun Hwan Kim, Hong-Jin Kim
Human papillomavirus (HPV) E6 protein is an oncoprotein with a pivotal role in cervical carcinogenesis. Expression and purification of HPV E6 from Escherichia coli (E. coli) has been difficult because of its strong hydrophobicity even when expressed as a fusion protein with glutathione S-transferase (GST). There has been no protocol suggested for purifying GST-tagged HPV E6 protein with high purity so far. Herein, we provide efficient protocol for purifying GST-HPV16 E6 protein for the first time. In the current study, the GST-tagged protein was expressed in E...
January 9, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28089880/a-combined-approach-for-enhancing-the-stability-of-recombinant-cis-dihydrodiol-naphthalene-dehydrogenase-from-pseudomonas-putida-g7-allowed-for-the-structural-and-kinetic-characterization-of-the-enzyme
#19
Débora Maria Abrantes Costa, Mariana Amalia Figueiredo Costa, Samuel Leite Guimarães, Juliana Barbosa Coitinho, Stefanya Velásquez Gómez, Tiago Antônio da Silva Brandão, Ronaldo Alves Pinto Nagem
The second enzyme of the naphthalene degradation pathway in Pseudomonas putida G7 is NahB, a dehydrogenase that converts cis-1,2-dihydroxy-1,2-dihydronaphthalene to 1,2-dihydroxynaphthalene. We report the cloning, optimization of expression, purification, kinetic studies and preliminary structural characterization of the recombinant NahB. The nahB gene was cloned into a T7 expression vector and the enzyme was overexpressed in Escherichia coli Rosetta (DE3) as an N-terminal hexa-histidine-tagged protein (6xHis-NahB)...
January 9, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28082061/purification-auto-activation-and-kinetic-characterization-of-apoptosis-signal-regulating-kinase-i
#20
John M Pleinis, Cameron W Davis, Caleb B Cantrell, David Y Qiu, Xuanzhi Zhan
Apoptosis signal-regulating kinase I (ASK1) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates the downstream MAP kinase kinases (MKKs) from two MAP kinase cascades: c-Jun N-terminal kinase (JNK) and p38. The essential physiological functions of ASK1 have attracted extensive attention. However, our understanding of the molecular mechanisms of ASK1, including the activation mechanism of ASK1 and the catalytic mechanism of ASK1-mediated MKK phosphorylation, remain unclear. The lack of purified ASK1 protein has hindered the elucidation of ASK1-initiated signal transduction mechanisms...
January 7, 2017: Protein Expression and Purification
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