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Protein expression and purification

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https://www.readbyqxmd.com/read/28220242/expression-of-clonorchis-sinensis-giiispla2-protein-in-baculovirus-infected-insect-cells-and-its-overexpression-facilitating-epithelial-mesenchymal-transition-in-huh7-cells-via-akt-pathway
#1
Mei Shang, Zhizhi Xie, Zeli Tang, Lei He, Xiaoyun Wang, Caiqin Wang, Yinjuan Wu, Ye Li, Lu Zhao, Zhiyue Lv, Zhongdao Wu, Yan Huang, Xinbing Yu, Xuerong Li
Although prior studies confirmed that group III secretory phospholipase A2 of Clonorchis sinensis (CsGIIIsPLA2) had stimulating effect on liver fibrosis by binding to LX-2 cells, large-scale expression of recombinant protein and its function in the progression of hepatoma are worth exploring. Because of high productivity and low lipopolysaccharides (LPS) in the Sf9-baculovirus expression system, we firstly used this system to express the coding region of CsGIIIsPLA2. The molecular weight of recombinant CsGIIIsPLA2 protein was about 34 kDa...
February 20, 2017: Parasitology Research
https://www.readbyqxmd.com/read/28218978/biochemical-characterization-of-metabolism-based-atrazine-resistance-in-amaranthus-tuberculatus-and-identification-of-an-expressed-gst-associated-with-resistance
#2
Anton F Evans, Sarah R O'Brien, Rong Ma, Aaron G Hager, Chance W Riggins, Kris N Lambert, Dean E Riechers
Rapid detoxification of atrazine in naturally tolerant crops such as maize (Zea mays) and grain sorghum (Sorghum bicolor) results from glutathione S-transferase (GST) activity. In previous research, two atrazine-resistant waterhemp (Amaranthus tuberculatus) populations from Illinois, U.S.A. (designated ACR and MCR) displayed rapid formation of atrazine-glutathione (GSH) conjugates, implicating elevated rates of metabolism as the resistance mechanism. Our main objective was to utilize protein purification combined with qualitative proteomics to investigate the hypothesis that enhanced atrazine detoxification, catalyzed by distinct GSTs, confers resistance in ACR and MCR...
February 20, 2017: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/28218648/expression-purification-and-monitoring-of-conformational-changes-of-hcb2-tmh67h8-in-different-membrane-mimetic-lipid-mixtures-using-circular-dichroism-and-nmr-techniques
#3
Elvis K Tiburu, Jianqin Zhuang, Heidimarie N A Fleischer, Patrick K Arthur, Gordon A Awandare
This work was intended to develop self-assembly lipids for incorporating G-protein coupled receptors (GPCRs) in order to improve the success rate for nuclear magnetic resonance spectroscopy (NMR) structural elucidation. We hereby report the expression and purification of uniformly (15)N-labeled human cannabinoid receptor-2 domain in insect cell media. The domain was refolded by screening several membrane mimetic environments. Different q ratios of isotropic bicelles were screened for solubilizing transmembrane helix 6, 7 and 8 (TMH67H8)...
February 17, 2017: Membranes
https://www.readbyqxmd.com/read/28216490/-expression-and-purification-of-foxo1-dna-binding-domain-and-its-dna-properties
#4
Yinuer Ha, Jun Li, Yongheng Chen, Lin Chen, Zhuchu Chen
To express and purify FOXO1 DNA binding domain (DBD) and to evaluate its DNA binding properties.
 Methods: Gene sequence of FOXO1-DBD was optimized and the recombinant strains were induced at low temperature to obtain soluble protein of FOXO1-DBD. Then, FOXO1-DBD protein was purified through Ni affinity chromatography and cation exchange. Finally, the DNA binding properties of FOXO1-DBD was evaluated by electrophoretic mobility shift assay (EMSA).
 Results: Most of soluble proteins were obtained by optimizing its genes sequence and induction at 21 ℃...
January 28, 2017: Zhong Nan da Xue Xue Bao. Yi Xue Ban, Journal of Central South University. Medical Sciences
https://www.readbyqxmd.com/read/28214589/enhanced-expression-and-purification-of-camelid-single-domain-vhh-antibodies-from-classical-inclusion-bodies
#5
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/28212428/a-novel-hemagglutinin-protein-produced-in-bacteria-protects-chickens-against-h5n1-highly-pathogenic-avian-influenza-viruses-by-inducing-h5-subtype-specific-neutralizing-antibodies
#6
Violetta Sączyńska, Agnieszka Romanik, Katarzyna Florys, Violetta Cecuda-Adamczewska, Małgorzata Kęsik-Brodacka, Krzysztof Śmietanka, Monika Olszewska, Katarzyna Domańska-Blicharz, Zenon Minta, Bogusław Szewczyk, Grażyna Płucienniczak, Andrzej Płucienniczak
The highly pathogenic (HP) H5N1 avian influenza viruses (AIVs) cause a mortality rate of up to 100% in infected chickens and pose a permanent pandemic threat. Attempts to obtain effective vaccines against H5N1 HPAIVs have focused on hemagglutinin (HA), an immunodominant viral antigen capable of eliciting neutralizing antibodies. The vast majority of vaccine projects have been performed using eukaryotic expression systems. In contrast, we used a bacterial expression system to produce vaccine HA protein (bacterial HA) according to our own design...
2017: PloS One
https://www.readbyqxmd.com/read/28211124/in-planta-expression-searching-for-the-genuine-chromophores-of-cryptochrome-3-from-arabidopsis-thaliana
#7
Wolfgang Gärtner
Göbel et al. present in this issue an exemplary study of identification of chromophores from Arabidopsis thaliana cryptochrome-3. Usually taken for granted, proteins and cofactors, respective chromophores, from heterologous expression are considered identical to material isolated from their genuine host. Cryptochromes carry two chromophores, an antenna cofactor and a functional flavin chromophore, both noncovalently embedded into the protein. In particular the antenna chromophore is loosely bound and often lost during protein purification...
January 2017: Photochemistry and Photobiology
https://www.readbyqxmd.com/read/28209207/recombinant-%C3%AE-actinin-subunit-antigens-of-trichomonas-vaginalis-as-potential-vaccine-candidates-in-protecting-against-trichomoniasis
#8
Yi-Ting Xie, Jiang-Mei Gao, Ya-Ping Wu, Petrus Tang, Geoff Hide, De-Hua Lai, Zhao-Rong Lun
BACKGROUND: Human trichomoniasis caused by Trichomonas vaginalis is one of the most common sexually transmitted diseases with more than 200 million cases worldwide. It has caused a series of health problems to patients. For prevention and control of infectious diseases, vaccines are usually considered as one of the most cost-efficient tools. However, until now, work on the development of T. vaginalis vaccines is still mainly focused on the screening of potential immunogens. Alpha-actinin characterized by high immunogenicity in T...
February 16, 2017: Parasites & Vectors
https://www.readbyqxmd.com/read/28205321/an-investigative-graduate-laboratory-course-for-teaching-modern-dna-techniques
#9
Alexandre de Lencastre, A Thomas Torello, Lani C Keller
This graduate-level DNA methods laboratory course is designed to model a discovery-based research project and engages students in both traditional DNA analysis methods and modern recombinant DNA cloning techniques. In the first part of the course, students clone the Drosophila ortholog of a human disease gene of their choosing using Gateway(®) cloning. In the second part of the course, students examine the expression of their gene of interest in human cell lines by reverse transcription PCR and learn how to analyze data from quantitative reverse transcription PCR (qRT-PCR) experiments...
February 16, 2017: Biochemistry and Molecular Biology Education
https://www.readbyqxmd.com/read/28202756/a-glycosylation-benchmark-profile-for-hiv-1-envelope-glycoprotein-production-based-on-eleven-env-trimers
#10
Eden P Go, Haitao Ding, Shijian Zhang, Rajesh P Ringe, Nathan Nicely, David Hua, Robert T Steinbock, Michael Golabek, James Alin, S Munir Alam, Albert Cupo, Barton F Haynes, John C Kappes, John P Moore, Joseph G Sodroski, Heather Desaire
HIV-1 envelope glycoprotein (Env) glycosylation is important because individual glycans are components of multiple broadly neutralizing antibody epitopes, while shielding other sites that might otherwise be immunogenic. The glycosylation on Env is influenced by a variety of factors, including the genotype of the protein, the cell line used for its expression, and the details of the construct design. Here, we used a mass spectrometry-based approach to map the complete glycosylation profile at every site in multiple HIV-1 Env trimers, accomplishing two goals: 1) We determined which glycosylation sites contain conserved glycan profiles across many trimeric Envs...
February 15, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28199087/expression-purification-and-properties-of-a-human-arachidonoyl-specific-isoform-of-diacylglycerol-kinase
#11
William Jennings, Sejal Doshi, Prasanta Kumar Hota, Aaron Prodeus, Stephanie Black, Richard M Epand
Diacylglycerol kinase epsilon (DGKε) catalyzes the phosphorylation of diacylglycerol producing phosphatidic acid. DGKε demonstrates exquisite specificity for the acyl chains of diacylglycerol. This contributes to the specificity of the PI-cycle for lipid intermediates containing particular acyl chains. Dysregulation of DGKε perturbs lipid signaling and biosynthesis, which has been linked to epilepsy, Huntington's disease and heart disease. Recessive loss-of-function mutations in the DGKε gene cause atypical hemolytic uremic syndrome...
February 15, 2017: Biochemistry
https://www.readbyqxmd.com/read/28198449/recombinant-vacuolar-iron-transporter-family-homologue-pfvit-from-human-malaria-causing-plasmodium-falciparum-is-a-fe-2-h-exchanger
#12
Paola Labarbuta, Katie Duckett, Catherine H Botting, Osama Chahrour, John Malone, John P Dalton, Christopher J Law
Vacuolar iron transporters (VITs) are a poorly understood family of integral membrane proteins that can function in iron homeostasis via sequestration of labile Fe(2+) into vacuolar compartments. Here we report on the heterologous overexpression and purification of PfVIT, a vacuolar iron transporter homologue from the human malaria-causing parasite Plasmodium falciparum. Use of synthetic, codon-optimised DNA enabled overexpression of functional PfVIT in the inner membrane of Escherichia coli which, in turn, conferred iron tolerance to the bacterial cells...
February 15, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28194527/impairment-of-infectious-laryngotracheitis-virus-replication-by-deletion-of-the-ul-1-gene
#13
M Nadimpalli, S W Lee, J M Devlin, J R Gilkerson, C A Hartley
Infectious laryngotracheitis virus (ILTV) encodes several unique genes, including a pair of unique nuclear proteins UL0 and UL[-1] that are expressed during replication in cell culture. Although the UL0 gene has been shown to be dispensable for replication, the role of UL[-1] has not been elucidated. In this study a deletion mutant of ILTV lacking the UL[-1] gene was constructed using homologous recombination. The coding sequences of the gene were replaced with the gene for enhanced green fluorescent protein and the cytomegalovirus major immediate early promoter element...
February 13, 2017: Archives of Virology
https://www.readbyqxmd.com/read/28193333/heterologous-expression-of-%C3%AE-glutamyl-transpeptidase-from-bacillus-atrophaeus-gs-16-and-its-application-in-the-synthesis-of-%C3%AE-d-glutamyl-l-tryptophan-a-known-immunomodulatory-peptide
#14
Meenu Saini, Shruti Bindal, Rani Gupta
Gamma-glutamyl transpeptidase from a mesophilic bacterium Bacillus atrophaeus GS-16 (BaGGT) was expressed heterologously in E. coli using pET-51b vector. Maximum production of BaGGT was obtained at 16°C after 16h of IPTG induction and the protein, in its native conformation, was active as a heterooctamer which was composed of four heterodimeric units combined together. One heterodimeric unit constituted two subunits with molecular masses of 45kDa and 21kDa, respectively. The recombinant enzyme was purified by one step His-tag affinity purification protocol with a specific activity of 90U/mg and 5...
April 2017: Enzyme and Microbial Technology
https://www.readbyqxmd.com/read/28192993/quantitative-affinity-determination-by-fluorescence-anisotropy-measurements-of-individual-nanoliter-droplets
#15
Fabrice Gielen, Maren Butz, Eric J Rees, Miklos Erdelyi, Tommaso Moschetti, Marko Hyvönen, Joshua B Edel, Clemens F Kaminski, Florian Hollfelder
Fluorescence anisotropy measurements of reagents compartmentalized into individual nanoliter droplets are shown to yield high-resolution binding curves from which precise dissociation constants (Kd) for protein-peptide interactions can be inferred. With the current platform, four titrations can be obtained per minute (based on ∼100 data points each), with stoichiometries spanning more than 2 orders of magnitude and requiring only tens of microliters of reagents. In addition to affinity measurements with purified components, Kd values for unpurified proteins in crude cell lysates can be obtained without prior knowledge of the concentration of the expressed protein, so that protein purification can be avoided...
January 17, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28192372/rna-binding-protein-pspc1-promotes-the-differentiation-dependent-nuclear-export-of-adipocyte-rnas
#16
Jiexin Wang, Prashant Rajbhandari, Andrey Damianov, Areum Han, Tamer Sallam, Hironori Waki, Claudio J Villanueva, Stephen D Lee, Ronni Nielsen, Susanne Mandrup, Karen Reue, Stephen G Young, Julian Whitelegge, Enrique Saez, Douglas L Black, Peter Tontonoz
A highly orchestrated gene expression program establishes the properties that define mature adipocytes, but the contribution of posttranscriptional factors to the adipocyte phenotype is poorly understood. Here we have shown that the RNA-binding protein PSPC1, a component of the paraspeckle complex, promotes adipogenesis in vitro and is important for mature adipocyte function in vivo. Cross-linking and immunoprecipitation followed by RNA sequencing revealed that PSPC1 binds to intronic and 3'-untranslated regions of a number of adipocyte RNAs, including the RNA encoding the transcriptional regulator EBF1...
February 13, 2017: Journal of Clinical Investigation
https://www.readbyqxmd.com/read/28188870/functional-expression-and-purification-of-recombinant-hepcidin25-production-in-escherichia-coli-using-sumo-fusion-technology
#17
Vahideh Sadr, Behnaz Saffar, Rahman Emamzadeh
Hepcidin25 is a small cysteine-rich peptide hormone known as a new class of antimicrobial peptides. The purpose of the present study was to express, purify and investigate the antibacterial properties of recombinant human hepcidin25 protein production in Escherichia coli. Human hepcidin25 gene was optimized and fused to a small ubiquitin-related modifier (SUMO) gene for higher expression. Then SUMO-hepcidin25 was cloned into the pET-32a (+) vector and expressed in E.coli Origami. The fusion protein with a molecular weight of approximately 35kDa was analyzed on SDS-PAGE gel...
February 7, 2017: Gene
https://www.readbyqxmd.com/read/28188527/parallel-exploration-of-interaction-space-by-bioid-and-affinity-purification-coupled-to-mass-spectrometry
#18
Geoffrey G Hesketh, Ji-Young Youn, Payman Samavarchi-Tehrani, Brian Raught, Anne-Claude Gingras
Complete understanding of cellular function requires knowledge of the composition and dynamics of protein interaction networks, the importance of which spans all molecular cell biology fields. Mass spectrometry-based proteomics approaches are instrumental in this process, with affinity purification coupled to mass spectrometry (AP-MS) now widely used for defining interaction landscapes. Traditional AP-MS methods are well suited to providing information regarding the temporal aspects of soluble protein-protein interactions, but the requirement to maintain protein-protein interactions during cell lysis and AP means that both weak-affinity interactions and spatial information is lost...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28188071/a-good-manufacturing-practice-grade-standard-protocol-for-exclusively-human-mesenchymal-stromal-cell-derived-extracellular-vesicles
#19
Karin Pachler, Thomas Lener, Doris Streif, Zsuzsanna A Dunai, Alexandre Desgeorges, Martina Feichtner, Michaela Öller, Katharina Schallmoser, Eva Rohde, Mario Gimona
BACKGROUND AIMS: Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) may contribute to biological processes such as tissue regeneration, immunomodulation and neuroprotection. Evaluation of their therapeutic potential and application in future clinical trials demands thorough characterization of EV content and production under defined medium conditions, devoid of xenogenic substances and serum-derived vesicles. Addressing the apparent need for such a growth medium, we have developed a medium formulation based on pooled human platelet lysate (pHPL), free from animal-derived xenogenic additives and depleted of EVs...
February 7, 2017: Cytotherapy
https://www.readbyqxmd.com/read/28187984/construction-of-a-viral-t2a-peptide-based-knock-in-mouse-model-for-enhanced-cre-recombinase-activity-and-fluorescent-labeling-of-podocytes
#20
Sybille Koehler, Sebastian Brähler, Fabian Braun, Henning Hagmann, Markus M Rinschen, Martin R Späth, Martin Höhne, F Thomas Wunderlich, Bernhard Schermer, Thomas Benzing, Paul T Brinkkoetter
Podocyte injury is a key event in glomerular disease leading to proteinuria and opening the path toward glomerular scarring. As a consequence, glomerular research strives to discover molecular mechanisms and signaling pathways affecting podocyte health. The hNphs2.Cre mouse model has been a valuable tool to manipulate podocyte-specific genes and to label podocytes for lineage tracing and purification. Here we designed a novel podocyte-specific tricistronic Cre mouse model combining codon improved Cre expression and fluorescent cell labeling with mTomato under the control of the endogenous Nphs2 promoter using viral T2A-peptides...
February 7, 2017: Kidney International
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