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Protein expression and purification

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https://www.readbyqxmd.com/read/28816641/preparation-of-recombinant-human-thymic-stromal-lymphopoietin-protein
#1
Yanlu Zhang, Liehua Wu, Jingai Yu, JianFeng Mei, Yu Yi, JianShu Chen, Guoqing Ying
Human thymic stromal lymphopoietin (hTSLP) protein plays a central role in inflammation. Characterizing properties of hTSLP requires a recombinant overexpression system that produces correctly folded, active hTSLP. In this report, an efficient overexpression system for production of hTSLP was developed. We constructed expression plasmids of the full-length hTslp gene with or without the signal peptide and transformed the plasmids into E. coli. The design of the recombinant proteins included an N-terminal His-tag, which facilitated purification...
August 17, 2017: Preparative Biochemistry & Biotechnology
https://www.readbyqxmd.com/read/28811266/expression-and-purification-of-a-difficult-sarcomeric-protein-telethonin
#2
Huanbo Tan, Wencheng Su, Pengju Wang, Wenyu Zhang, Michael Sattler, Peijian Zou
Telethonin anchors the N-terminal region of titin in the Z-disk of the sarcomere by binding to two immunoglobulin-like (Ig) domains (Z1 and Z2) of titin (Z1Z2). Thereby telethonin plays an important role in myofibril assembly and in muscle development and functional regulation. The expression and purification of recombinant telethonin is very challenging. In previous studies, recombinant telethonin expressed from E. coli was refolded in the presence of Z1Z2. Here, we report various strategies to establish a reliable and efficient protocol for the preparation of telethonin and titin Z1Z2 protein...
August 12, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28811070/evaluation-of-sequence-variability-in-hiv-1-gp41-c-peptide-helix-grafted-proteins
#3
Rachel L Tennyson, Susanne N Walker, Terumasa Ikeda, Reuben S Harris, Brian R McNaughton
Many therapeutically-relevant protein-protein interactions (PPIs) have been reported that feature a helix and helix-binding cleft at the interface. Given this, different approaches to disrupting such PPIs have been developed. While short peptides (<15 amino acids) typically do not fold into a stable helix, researchers have reported chemical approaches to constraining helix structure. However, these approaches rely on laborious, and often expensive, chemical synthesis and purification. Our premise is that protein-based solutions that stabilize a therapeutically-relevant helix offer a number of advantages...
August 1, 2017: Bioorganic & Medicinal Chemistry
https://www.readbyqxmd.com/read/28808967/purification-of-cyclic-gmp-amp-from-viruses-and-measurement-of-its-activity-in-cell-culture
#4
Alice Mayer, Jonathan Maelfait, Anne Bridgeman, Jan Rehwinkel
Sensing of cytoplasmic DNA by cGAS is essential for the initiation of immune responses against several viruses. cGAS also plays important roles in some autoinflammatory and autoimmune diseases and may be involved in immune responses targeting cancer cells. Once activated, cGAS catalyzes the formation of the di-nucleotide 2'-3'-cyclic GMP-AMP (cGAMP), which propagates a signaling cascade leading to the production of type I interferons (IFNs). Interestingly, cGAMP is incorporated into enveloped viruses and is transferred to newly infected cells by virions...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28808944/effect-of-ha330-resin-directed-hemoadsorption-on-a-porcine-acute-respiratory-distress-syndrome-model
#5
Xuefeng Xu, Chune Jia, Sa Luo, Yanming Li, Fei Xiao, Huaping Dai, Chen Wang
BACKGROUND: Blood purification is an emerging approach to dampening the cytokine storm. This study aims to assess the efficacy of HA330 resin-directed hemoadsorption (HA) on endotoxin-induced porcine acute respiratory distress syndrome (ARDS) model. METHODS: Twenty-four Chinese domestic pigs were allocated into saline group receiving intravenous infusion of saline (N = 6) and endotoxin group receiving intravenous infusion of LPS (N = 18). When ALI model was initially diagnosed, six pigs in the LPS and saline group were killed for BALF and histopathological analysis...
August 14, 2017: Annals of Intensive Care
https://www.readbyqxmd.com/read/28807316/vaccination-with-eimeria-tenella-elongation-factor-1%C3%AE-recombinant-protein-induces-protective-immunity-against-e-tenella-and-e-maxima-infections
#6
Rui-Qing Lin, Hyun S Lillehoj, Seung Kyoo Lee, Sungtaek Oh, Alfredo Panebra, Erik P Lillehoj
Avian coccidiosis is caused by multiple species of the apicomplexan protozoan, Eimeria, and is one of the most economically devastating enteric diseases for the poultry industry worldwide. Host immunity to Eimeria infection, however, is relatively species-specific. The ability to immunize chickens against different species of Eimeria using a single vaccine will have a major beneficial impact on commercial poultry production. In this paper, we describe the molecular cloning, purification, and vaccination efficacy of a novel Eimeria vaccine candidate, elongation factor-1α (EF-1α)...
August 30, 2017: Veterinary Parasitology
https://www.readbyqxmd.com/read/28806153/expression-purification-and-refolding-of-human-lipocalin-6-and-production-of-a-monoclonal-antibody-against-this-protein
#7
Jiong Chen, Wei Feng, Yue Zhao, Yaqin Li, Fei Zhan
Human lipocalin 6 (hLCN6) is a member of the lipocalin family, which is a group of structurally conserved hydrophobic ligand binding proteins, and widely distributed in animal, plant, and bacteria. Specific expression of hLCN6 in the epididymis and localization of this protein on the surface of spermatozoa suggest a role played by hLCN6, which may function as a transporter to carry ligands in the epididymal channel. However, the role of hLCN6 in sperm maturation has been largely unknown due to the lack of effective antibodies...
August 2017: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
https://www.readbyqxmd.com/read/28805269/cloning-and-bacterial-expression-systems-for-recombinant-human-heparanase-production-substrate-specificity-investigation-by-docking-of-a-putative-heparanase-substrate
#8
Angela Pennacchio, Alessandro Capo, Simonetta Caira, Annabella Tramice, Antonio Varriale, Maria Staiano, Sabato D'Auria
Human heparanase (HPSE) is an enzyme that degrades the extracellular matrix. It is implicated in a multiplicity of physiological and pathological processes encouraging angiogenesis and tumor metastasis. The protein is a heterodimer composed from a subunit of 8 kDa and a subunit of 50 kDa. The two protein subunits are non-covalently associated. The cloning and expression of the two protein subunits in Escherichia coli, and their subsequent purification to homogeneity under native conditions result in the production of an active heparanase enzyme...
August 14, 2017: Biotechnology and Applied Biochemistry
https://www.readbyqxmd.com/read/28803540/preparation-of-highly-multiplexed-small-rna-sequencing-libraries
#9
Helena Persson, Rolf Søkilde, Anna Chiara Pirona, Carlos Rovira
MicroRNAs (miRNAs) are ~22-nucleotide-long small non-coding RNAs that regulate the expression of protein-coding genes by base pairing to partially complementary target sites, preferentially located in the 3´ untranslated region (UTR) of target mRNAs. The expression and function of miRNAs have been extensively studied in human disease, as well as the possibility of using these molecules as biomarkers for prognostication and treatment guidance. To identify and validate miRNAs as biomarkers, their expression must be screened in large collections of patient samples...
August 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28802994/expression-and-purification-of-norovirus-virus-like-particles-in-escherichia-coli-and-their-immunogenicity-in-mice
#10
Yuqi Huo, Xin Wan, Tong Ling, Jie Wu, Wenhui Wang, Shuo Shen
Norovirus (NoV) virus like particles (VLPs) produced in Spodoptera frugiperda (Sf9) cells have been tested in human volunteers as vaccine candidate and were shown to be protective against NoV induced acute gastroenteritis. In this study, prevailing Sydney-2012-like NoV major capsid protein gene with or without N-terminal deletions (N26 and N38, 26 and 38 amino acids deleted from N terminus,respectively) were sub-cloned into prokaryotic expression vector, pCold III and pCold IV. Soluble and insoluble proteins were detected for both vectors after induction and higher levels of protein expression were observed for constructs pCold III-N26 and pCold III-N38...
August 9, 2017: Molecular Immunology
https://www.readbyqxmd.com/read/28802578/purification-of-functional-reprogramming-factors-in-mammalian-cell-using-flag-tag
#11
Min-Joon Han, HaRam Kim, Carla O'Reilly, Chun-Hyung Kim
Induced pluripotent stem cells (iPSCs) technology is a method for generating pluripotent stem cells in vitro from fully differentiated cells such as fibroblast cells. The potential applications of iPSC technology in cell therapy and disease modeling could influence current medical practices. Despite current advances in iPSC technology, many patient-derived reprogrammed cells are not suitable for clinical trial because most protocols rely on virus-based techniques, which pose the risk of integration of the viral genome into the chromosomes...
August 9, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/28801830/cloning-and-expression-analysis-of-human-amelogenin-in-nicotiana-benthamiana-plants-by-means-of-a-transient-expression-system
#12
Mattia Pegoraro, Slavica Matić, Barbara Pergolizzi, Luca Iannarelli, Andrea M Rossi, Marco Morra, Emanuela Noris
Enamel is the covering tissue of teeth, made of regularly arranged hydroxyapatite crystals deposited on an organic matrix composed of 90% amelogenin that is completely degraded at the end of the enamel formation process. Amelogenin has a biomineralizing activity, forming nanoparticles or nanoribbons that guide hydroxyapatite deposit, and regenerative functions in bone and vascular tissue and in wound healing. Biotechnological products containing amelogenin seem to facilitate these processes. Here, we describe the production of human amelogenin in plants by transient transformation of Nicotiana benthamiana with constructs carrying synthetic genes with optimized human or plant codons...
August 11, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28801725/optimization-of-soluble-expression-and-purification-of-recombinant-human-rhinovirus-type-14-3c-protease-using-statistically-designed-experiments-isolation-and-characterization-of-the-enzyme
#13
Georgia Antoniou, Irineos Papakyriacou, Christos Papaneophytou
Human rhinovirus (HRV) 3C protease is widely used in recombinant protein production for various applications such as biochemical characterization and structural biology projects to separate recombinant fusion proteins from their affinity tags in order to prevent interference between these tags and the target proteins. Herein, we report the optimization of expression and purification conditions of glutathione S-transferase (GST)-tagged HRV 3C protease by statistically designed experiments. Soluble expression of GST-HRV 3C protease was initially optimized by response surface methodology (RSM), and a 5...
August 11, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28800364/expression-optimization-of-a-cell-membrane-penetrating-human-papillomavirus-type-16-therapeutic-vaccine-candidate-in-nicotiana-benthamiana
#14
Romana J R Yanez, Renate Lamprecht, Milaid Granadillo, Brandon Weber, Isis Torrens, Edward P Rybicki, Inga I Hitzeroth
High-risk human papillomaviruses (hr-HPVs) cause cervical cancer, the fourth most common cancer in women worldwide. A HPV-16 candidate therapeutic vaccine, LALF32-51-E7, was developed by fusing a modified E7 protein to a bacterial cell-penetrating peptide (LALF): this elicited both tumour protection and regression in pre-clinical immunization studies. In the current study, we investigated the potential for producing LALF32-51-E7 in a plant expression system by evaluating the effect of subcellular localization and usage of different expression vectors and gene silencing suppressors...
2017: PloS One
https://www.readbyqxmd.com/read/28794925/expression-of-codon-optimized-tgmic16-in-three-escherichia-coli-strains
#15
Hui Sun, Jin Li, Gong-Zhen Liu, Kun Yin, Yong Cui, Ting Xiao, Chao Xu, Bing-Cheng Huang, Qing-Kuan Wei
In a previous study, we found that rabbit anti-Toxoplasma gondii serum was capable of recognizing truncated T. gondii microneme protein 16 (TgMIC16), indicating that TgMIC16 is an essential antigenic T. gondii protein. However, the broad application of this recombinant protein is limited by its low expression level. In this study, we performed codon optimization of TgMIC16 by changing the codon-adaptation index from 0.22 to 1.0 without altering the amino acid sequence and expressed the optimized gene in three different Escherichia coli strains, followed by comparison of soluble recombinant-protein expression and yield...
August 2017: 3 Biotech
https://www.readbyqxmd.com/read/28791907/a-one-step-procedure-for-immobilising-the-thermostable-carbonic-anhydrase-sspca-on-the-surface-membrane-of-escherichia-coli
#16
Sonia Del Prete, Rosa Perfetto, Mosè Rossi, Fatmah A S Alasmary, Sameh M Osman, Zeid AlOthman, Claudiu T Supuran, Clemente Capasso
The carbonic anhydrase superfamily (CA, EC 4.2.1.1) of metalloenzymes is present in all three domains of life (Eubacteria, Archaea, and Eukarya), being an interesting example of convergent/divergent evolution, with its seven families (α-, β-, γ-, δ-, ζ-, η-, and θ-CAs) described so far. CAs catalyse the simple, but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons. Recently, our groups characterised the α-CA from the thermophilic bacterium, Sulfurihydrogenibium yellowstonense finding a very high catalytic activity for the CO2 hydration reaction (kcat = 9...
December 2017: Journal of Enzyme Inhibition and Medicinal Chemistry
https://www.readbyqxmd.com/read/28791342/generation-of-a-novel-trail-mutant-by-proline-to-arginine-substitution-based-on-codon-bias-and-its-antitumor-effects
#17
Aijing Zhu, Xiuyun Wang, Min Huang, Chen Chen, Juan Yan, Qi Xu, Lijia Wei, Xianzhou Huang, Hong Zhu, Cheng Yi
TNF ligand superfamily member 10 (TRAIL) is a member of the tumor necrosis factor superfamily. The present study was performed in an effort to increase the expression of soluble (s)TRAIL by rebuilding the gene sequence of TRAIL. Three principles based on the codon bias of Escherichia coli were put forward to design the rebuild strategy. Relying on these three principles, a P7R mutation near the N‑terminal region of sTRAIL, named TRAIL‑Mu, was designed. TRAIL‑Mu was subsequently cloned into the PTWIN1 plasmid and expressed in E...
August 2, 2017: Molecular Medicine Reports
https://www.readbyqxmd.com/read/28789852/recombinant-calponin-of-human-filariid-brugia-malayi-secondary-structure-and-immunoprophylactic-potential
#18
Shiv K Verma, Ashish Arora, P Kalpana Murthy
In the search for potential vaccine candidates for the control of human lymphatic filariasis, we recently identified calponin-like protein, that regulates actin/myosin interactions, in a proinflammatory fraction F8 (45.24-48.64kDa) of Brugia malayi adult worms. In the present study, the gene was cloned, expressed, and the recombinant Calponin of B. malayi (r-ClpBm) was prepared and characterized. r-ClpBm bears homology with OV9M of Onchocerca volvulus, a non-lymphatic filariid that causes loss of vision and cutaneous pathology...
August 5, 2017: Vaccine
https://www.readbyqxmd.com/read/28789570/an-overview-of-bacillus-proteases-from-production-to-application
#19
Fabiano Jares Contesini, Ricardo Rodrigues de Melo, Hélia Harumi Sato
Proteases have a broad range of applications in industrial processes and products and are representative of most worldwide enzyme sales. The genus Bacillus is probably the most important bacterial source of proteases and is capable of producing high yields of neutral and alkaline proteolytic enzymes with remarkable properties, such as high stability towards extreme temperatures, pH, organic solvents, detergents and oxidizing compounds. Therefore, several strategies have been developed for the cost-effective production of Bacillus proteases, including optimization of the fermentation parameters...
August 8, 2017: Critical Reviews in Biotechnology
https://www.readbyqxmd.com/read/28783894/expression-and-purification-of-transmembrane-protein-mere-from-mercury-resistant-bacillus-cereus
#20
Aatif Amin, Zakia Latif, Stanley J Opella
Mercury resistant (Hg(R)) bacteria were isolated from heavy metal polluted wastewater and soil collected near to tanneries of district Kasur, Pakistan. Bacterial isolates AZ-1, AZ-2 and AZ-3 showed resistance up to 40 μg/mL against mercuric chloride (HgCl2). 16S rDNA ribotyping and phylogenetic analysis were performed for the characterization of selected isolates as Bacillus sp. AZ-1 (KT270477), Bacillus cereus AZ-2 (KT270478) and Bacillus cereus AZ-3 (KT270479). Phylogenetic relationship on the basis of merA nucleotide sequence confirmed 51-100% homology with the corresponding region of the merA gene of already reported mercury resistant Gram positive bacteria...
August 8, 2017: Journal of Microbiology and Biotechnology
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