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Protein expression and purification

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https://www.readbyqxmd.com/read/28100961/auxin-extraction-and-purification-based-on-recombinant-aux-iaa-proteins
#1
Yi Su, Weigui Luo, Xiaofei Chen, Huizhen Liu, Yueqing Hu, Wanhuang Lin, Langtao Xiao
BACKGROUND: Indole-3-acetic acid (IAA) extraction and purification are of great importance in auxin research, which is a hot topic in the plant growth and development field. Solid-phase extraction (SPE) is frequently used for IAA extraction and purification. However, no IAA-specific SPE columns are commercially available at the moment. Therefore, the development of IAA-specific recognition materials and IAA extraction and purification methods will help researchers meet the need for more precise analytical methods for research on phytohormones...
2017: Biological Procedures Online
https://www.readbyqxmd.com/read/28098337/characterisation-of-the-enzymatic-properties-of-mpapr1-an-aspartic-protease-secreted-by-the-wine-yeast-metschnikowia-pulcherrima
#2
Louwrens Wiid Theron, Marina Bely, Benoit Divol
BACKGROUND: MpAPr1, encoding an acid protease from the wine yeast Metschnikowia pulcherrima IWBT Y1123, was previously isolated and shown to display potential activity against casein and grape proteins. However, its characterisation remained partial. RESULTS: MpAPr1 was cloned into the pGAPZαA vector and transformed into Komagataella pastoris X33 for heterologous expression. After verification of activity, the enzyme properties were characterised. Protease activity within the concentrated supernatant was retained over a pH range of 3...
January 18, 2017: Journal of the Science of Food and Agriculture
https://www.readbyqxmd.com/read/28098257/a-comprehensive-platform-for-the-analysis-of-ubiquitin-like-protein-modifications-using-in-vivo-biotinylation
#3
Lucia Pirone, Wendy Xolalpa, Jón Otti Sigurðsson, Juanma Ramirez, Coralia Pérez, Monika González, Ainara Ruiz de Sabando, Félix Elortza, Manuel S Rodriguez, Ugo Mayor, Jesper V Olsen, Rosa Barrio, James D Sutherland
Post-translational modification by ubiquitin and ubiquitin-like proteins (UbLs) is fundamental for maintaining protein homeostasis. Efficient isolation of UbL conjugates is hampered by multiple factors, including cost and specificity of reagents, removal of UbLs by proteases, distinguishing UbL conjugates from interactors, and low quantities of modified substrates. Here we describe bioUbLs, a comprehensive set of tools for studying modifications in Drosophila and mammals, based on multicistronic expression and in vivo biotinylation using the E...
January 18, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28093085/gene-design-fusion-technology-and-tev-cleavage-conditions-influence-the-purification-of-oxidized-disulphide-rich-venom-peptides-in-escherichia-coli
#4
Ana Filipa Sequeira, Jeremy Turchetto, Natalie J Saez, Fanny Peysson, Laurie Ramond, Yoan Duhoo, Marilyne Blémont, Vânia O Fernandes, Luís T Gama, Luís M A Ferreira, Catarina I P I Guerreiro, Nicolas Gilles, Hervé Darbon, Carlos M G A Fontes, Renaud Vincentelli
BACKGROUND: Animal venoms are large, complex libraries of bioactive, disulphide-rich peptides. These peptides, and their novel biological activities, are of increasing pharmacological and therapeutic importance. However, recombinant expression of venom peptides in Escherichia coli remains difficult due to the significant number of cysteine residues requiring effective post-translational processing. There is also an urgent need to develop high-throughput recombinant protocols applicable to the production of reticulated peptides to enable efficient screening of their drug potential...
January 17, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/28092034/expression-and-purification-of-sh2-domains-using-baculovirus-expression-system
#5
Mari Ogiue-Ikeda, Kazuya Machida
Recombinant proteins expressed in bacteria are sometimes insoluble, aggregated, and incorrectly folded. For those Src homology 2 (SH2) domains that are insoluble in bacteria, baculovirus-insect cell expression systems can be an alternative to produce soluble and functionally active proteins. We describe a protocol for cloning and purification of GST-tagged SH2 domains using the Bac-to-Bac baculovirus expression system.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28092032/expression-and-purification-of-soluble-stat5b-stat3-proteins-for-sh2-domain-binding-assay
#6
Akira Asai, Kazuyuki Takakuma
When a large hydrophobic full-length protein is expressed in bacteria, it is often challenging to obtain recombinant proteins in the soluble fraction. One way to overcome this challenge is expression of deletion mutants that have improved solubility while maintaining biological activity. In this chapter, we describe a protocol for expression of truncated forms of STAT5b and STAT3 proteins that are soluble and retain SH2-mediated activity for phospho-Tyr peptide recognition.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28092031/expression-and-production-of-sh2-domain-proteins
#7
Bernard A Liu, Mari Ogiue-Ikeda, Kazuya Machida
The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating signaling events downstream of receptor tyrosine kinases (RTKs), adaptors, and scaffolds. Over a hundred SH2 domains are present in mammals, each having a unique specificity which determines its interactions with multiple binding partners. One of the essential tools necessary for studying and determining the role of SH2 domains in phosphotyrosine signaling is a set of soluble recombinant SH2 proteins. Here we describe methods, based on a broad experience with purification of all SH2 domains, for the production of SH2 domain proteins needed for proteomic and biochemical-based studies such as peptide arrays, mass-spectrometry, protein microarrays, reverse-phase microarrays, and high-throughput fluorescence polarization (HTP-FP)...
2017: Methods in Molecular Biology
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
#8
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
#9
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/28088197/production-of-human-pro-relaxin-h2-in-the-yeast-pichia-pastoris
#10
D Cimini, K Della Corte, R Finamore, L Andreozzi, A Stellavato, A V A Pirozzi, F Ferrara, R Formisano, M De Rosa, M Chino, L Lista, A Lombardi, V Pavone, C Schiraldi
BACKGROUND: Initially known as the reproductive hormone, relaxin was shown to possess other therapeutically useful properties that include extracellular matrix remodeling, anti-inflammatory, anti-ischemic and angiogenic effects. All these findings make relaxin a potential drug for diverse medical applications. Its precursor, pro-relaxin, is an 18 kDa protein, that shows activity in in vitro assays. Since extraction of relaxin from animal tissues raises several issues, prokaryotes and eukaryotes were both used as expression systems for recombinant relaxin production...
January 14, 2017: BMC Biotechnology
https://www.readbyqxmd.com/read/28087367/production-of-recombinant-proteins-in-escherichia-coli-tagged-with-the-fusion-protein-cusf3h
#11
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 10, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28077636/klebsiella-phage-%C3%AE-k64-1-encodes-multiple-depolymerases-for-multiple-host-capsular-types
#12
Yi-Jiun Pan, Tzu-Lung Lin, Ching-Ching Chen, Yun-Ting Tsai, Yi-Hsiang Cheng, Yi-Yin Chen, Pei-Fang Hsieh, Yi-Tsung Lin, Jin-Town Wang
: The genome of the multi-host bacteriophage ΦK64-1, capable of infecting Klebsiella capsular types K1, K11, K21, K25, K30, K35, K64, and K69, as well as new capsular types KN4 and KN5 was analyzed and revealed that 11 genes (S1-1, S1-2, S1-3, S2-1, S2-2, S2-3, S2-4, S2-5, S2-6, S2-7, and S2-8) encode proteins with amino acid sequence similarity to tail fibers/spikes or lyases. S2-5 previously was shown to encode a K64 capsule depolymerase (K64dep). Specific capsule-degrading activities of an additional 8 putative capsule depolymerases (S2-4 against K1; S1-1 against K11; S1-3 against K21; S2-2 against K25; S2-6 against K30/K69; S2-3 against K35; S1-2 against KN4; S2-1 against KN5) was demonstrated by expression and purification of the recombinant proteins...
January 11, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28077445/human-antiviral-protein-ifix-suppresses-viral-gene-expression-during-hsv-1-infection-and-is-counteracted-by-virus-induced-proteasomal-degradation
#13
Marni S Crow, Ileana M Cristea
The interferon inducible protein X (IFIX), a member of the PYHIN family, was recently recognized as an antiviral factor against infection with herpes simplex virus 1 (HSV-1). IFIX binds viral DNA upon infection and promotes antiviral cytokines expression. How IFIX exerts its host defense functions and whether it is inhibited by the virus remains unknown. Here, we integrated live cell microscopy, proteomics, IFIX domain characterization, and molecular virology to investigate IFIX regulation and antiviral functions during HSV-1 infection...
January 11, 2017: Molecular & Cellular Proteomics: MCP
https://www.readbyqxmd.com/read/28077248/alternative-splicing-at-exon-2-results-in-the-loss-of-the-catalytic-activity-of-mouse-dna-polymerase-iota-in-vitro
#14
Konstantin Y Kazachenko, Nataliya A Miropolskaya, Leonid V Gening, Vyacheslav Z Tarantul, Alena V Makarova
Y-family DNA polymerase iota (Pol ι) possesses both DNA polymerase and dRP lyase activities and was suggested to be involved in DNA translesion synthesis and base excision repair in mammals. The 129 strain of mice and its derivatives have a natural nonsense codon mutation in the second exon of the Pol ι gene resulting in truncation of the Pol ι protein. These mice were widely used as a Pol ι-null model for in vivo studies of the Pol ι function. However whether 129-derived strains of mice are fully deficient in the Pol ι functions was a subject of discussion since Pol ι mRNA undergoes alternative splicing at exon 2...
January 4, 2017: DNA Repair
https://www.readbyqxmd.com/read/28070540/data-on-the-optimizations-of-expression-and-purification-of-human-bip-grp78-protein-in-escherichia-coli
#15
Jiao Yang, Lei Zhou, Qinglian Liu
Human BiP/GRP78 is involved in the folding and assembly of proteins in the endoplasmic reticulum. The proteins for crystallization in good amount and quality are prerequisites for obtaining ideal crystals. To meet these requirements, different BiP/GRP78 constructs, competent cells, vectors, and concentrations of inducer were tested in order to obtain soluble BiP/GRP78 protein with the highest amount and best purity. The BiP-T229A-L3,4'-Smt3 fusion protein was expressed in a soluble manner and finally purified with the highest purity using size exclusion chromatography, which was suitable for further protein crystallization...
February 2017: Data in Brief
https://www.readbyqxmd.com/read/28068333/bdf1-bromodomains-are-essential-for-meiosis-and-the-expression-of-meiotic-specific-genes
#16
Encar García-Oliver, Claire Ramus, Jonathan Perot, Marie Arlotto, Morgane Champleboux, Flore Mietton, Christophe Battail, Anne Boland, Jean-François Deleuze, Myriam Ferro, Yohann Couté, Jérôme Govin
Bromodomain and Extra-terminal motif (BET) proteins play a central role in transcription regulation and chromatin signalling pathways. They are present in unicellular eukaryotes and in this study, the role of the BET protein Bdf1 has been explored in Saccharomyces cerevisiae. Mutation of Bdf1 bromodomains revealed defects on both the formation of spores and the meiotic progression, blocking cells at the exit from prophase, before the first meiotic division. This phenotype is associated with a massive deregulation of the transcription of meiotic genes and Bdf1 bromodomains are required for appropriate expression of the key meiotic transcription factor NDT80 and almost all the Ndt80-inducible genes, including APC complex components...
January 9, 2017: PLoS Genetics
https://www.readbyqxmd.com/read/28065867/cloning-expression-purification-and-biophysical-analysis-of-two-putative-halogenases-from-the-glycopeptide-a47-934-gene-cluster-of-streptomyces-toyocaensis
#17
Tabata P Cardoso, Larissa A de Sá, Priscila Dos S Bury, Sair M Chavez-Pacheco, Marcio V B Dias
Glycopeptides are an important class of antibiotics used in the treatment of several infections, including those caused by methicillin resistant Staphylococcus aureus. Glycopeptides are biosynthesized by a Non Ribosomal Peptide Synthase (NRPS) and the resulting peptide precursors are decorated by several tailoring enzymes, such as halogenases and glycosyltransferases. These enzymes are important targets of protein engineering to produce new derivatives of known antibiotics. Herein we show the production of two putative halogenases, denominated StaI and StaK, involved in the biosynthesis of the glycopeptide A47,934 in Streptomyces toyocaensis NRRL 15,009...
January 5, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28065274/probing-the-activity-of-eukaryotic-rhomboid-proteases-in-vitro
#18
B Cordier, M K Lemberg
Proteolysis within the membrane is a recent concept in biology. Rhomboid intramembrane serine proteases are conserved in evolution and serve as key switches in diverse cellular pathways ranging from signaling to protein degradation. Since deregulation of intramembrane proteolysis can lead to severe diseases including neurodegenerative disorders, dissecting their enzymatic function and specificity becomes crucial. As membrane proteins, their solubilization, and purification are technically challenging. As a start point for a comprehensive in vitro characterization of eukaryotic rhomboid proteases, we depict in this chapter a robust workflow to find the best conditions to obtain pure and active enzymes from a bacterial expression system...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28065273/screening-and-characterization-strategies-for-nanobodies-targeting-membrane-proteins
#19
S Veugelen, M Dewilde, B De Strooper, L Chávez-Gutiérrez
The study of membrane protein function and structure requires their successful detection, expression, solubilization, and/or reconstitution, which poses a challenging task and relies on the availability of suitable tools. Several research groups have successfully applied Nanobodies in the purification, as well as the functional and structural characterization of membrane proteins. Nanobodies are small, single-chain antibody fragments originating from camelids presenting on average a longer CDR3 which enables them to bind in cavities and clefts (such as active and allosteric sites)...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28065266/production-of-recombinant-rhomboid-proteases
#20
E Arutyunova, R Panigrahi, K Strisovsky, M J Lemieux
Rhomboid proteases are intramembrane enzymes that hydrolyze peptide bonds of transmembrane proteins in the lipid bilayer. They play a variety of roles in key biological events and are linked to several disease states. Over the last decade a great deal of structural and functional knowledge has been generated on this fascinating class of proteases. Both structural and kinetic analyses require milligram amounts of protein, which may be challenging for membrane proteins such as rhomboids. Here, we present a detailed protocol for optimization of expression and purification of three rhomboid proteases from Escherichia coli (ecGlpG), Haemophilus influenzae (hiGlpG), and Providencia stuartii (AarA)...
2017: Methods in Enzymology
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