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cell free protein synthesis

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https://www.readbyqxmd.com/read/28922469/one-pot-synthesis-of-gdp-mannose-by-a-multi-enzyme-cascade-for-enzymatic-assembly-of-lipid-linked-oligosaccharides
#1
Thomas F T Rexer, Anna Schildbach, Jan Klapproth, Angelika Schierhorn, Reza Mahour, Markus Pietzsch, Erdmann Rapp, Udo Reichl
Glycosylation of proteins is a key function of the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Glycosylated proteins play a crucial role in cell trafficking and signaling, cell-cell adhesion, blood-group antigenicity, and immune response. In addition, the glycosylation of proteins is an important parameter in the optimization of many glycoprotein-based drugs such as monoclonal antibodies. In vitro glycoengineering of proteins requires glycosyltransferases as well as expensive nucleotide sugars...
September 18, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28921431/cell-free-production-of-protein-biologics-within-24-h
#2
Challise J Sullivan, Erik D Pendleton, John Dresios
Protein biologics have emerged as a safe and effective group of drug products that can be used in a variety of medical disorders and clinical settings, including treatment of orphan diseases, personalized medicine, and point-of-care applications. However, the full potential of protein biologics for such applications will not be realized until there are methods available for rapid and cost-effective production of small scale products for individual needs. Here, we describe a modular and scalable method for rapid and adaptable production of protein-based medical products at small doses...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28919741/extracellular-biosynthesis-of-silver-nanoparticles-using-the-cell-free-filtrate-of-nematophagous-fungus-duddingtonia-flagrans
#3
Laryssa Pinheiro Costa Silva, Jairo Pinto Oliveira, Wanderson Juvencio Keijok, André Romero da Silva, Anderson Rocha Aguiar, Marco Cesar Cunegundes Guimarães, Carolina Magri Ferraz, Jackson Victor Araújo, Fernando Luiz Tobias, Fábio Ribeiro Braga
The biosynthesis of metallic nanoparticles (NPs) using biological systems such as fungi has evolved to become an important area of nanobiotechnology. Herein, we report for the first time the extracellular synthesis of highly stable silver NPs (AgNPs) using the nematophagous fungus Duddingtonia flagrans (AC001). The fungal cell-free filtrate was analyzed by the Bradford method and 3,5-dinitrosalicylic acid assay and used to synthesize the AgNPs in the presence of a 1 mM AgNO3 solution. They have been characterized by UV-Vis spectroscopy, X-ray diffraction, transmission electron microscopy, dynamic light scattering, Zeta potential measurements, Fourier-transform infrared, and Raman spectroscopes...
2017: International Journal of Nanomedicine
https://www.readbyqxmd.com/read/28918543/dietary-docosahexaenoic-acid-decreased-lipid-accumulation-via-inducing-adipocytes-apoptosis-of-grass-carp-ctenopharygodon-idella
#4
Ai Jin, Cai-Xia Lei, Jing-Jing Tian, Jian Sun, Hong Ji
The purpose of this study was to explore the mechanism of by which docosahexaenoic acid (DHA) inhibit the accumulation of adipose tissue lipid in grass carp (Ctenopharyngodon idella). We therefore designed two semi-purified diets, namely DHA-free (control) and DHA-supplemented, and fed them to grass carp (22.19 ± 1.76 g) for 3 and 6 weeks. DHA supplementation led to a significantly lower intraperitoneal fat index (IPFI) than that in the control group by reducing the number of adipocytes but significantly higher adipocyte size (P < 0...
September 17, 2017: Fish Physiology and Biochemistry
https://www.readbyqxmd.com/read/28918092/growth-rate-dependent-regulation-of-trna-level-and-charging-in-bacillus-licheniformis
#5
Iolanda Ferro, Klaus Liebeton, Zoya Ignatova
Cellular growth crucially depends on protein synthesis and the abundance of translational components. Among them, aminoacyl-tRNAs play a central role in biosynthesis and shape the kinetics of mRNA translation, thus influencing protein production. Here, we used microarray-based approaches to determine the charging levels and tRNA abundance of Bacillus licheniformis. We observed an interesting cross-talk among tRNA expression, charging pattern and growth rate. For a large subset of tRNAs, we found a co-regulated and augmented expression at high growth rate...
September 13, 2017: Journal of Molecular Biology
https://www.readbyqxmd.com/read/28917756/facile-synthesis-of-copper-ii-decorated-functional-mesoporous-material-for-specific-adsorption-of-histidine-rich-proteins
#6
Jiaojiao Wang, Rui Zhang, Xiaxia Yang, Xiaoyan Liu, Haixia Zhang
The Cu(2+)-decorated functional mesoporous material was fabricated by thermally initiated free-radical polymerization of octavinyl polyhedral oligomeric silsesquioxane. It was used as adsorbent for highly specific separation of histidine (His)-rich proteins in blood and cell lysate based on immobilized metal affinity chromatography. The functional mesoporous material (named as PPOSS-IDA-Cu(2+)) was characterized in detail and its selectivity and binding capacity were evaluated using a His-rich protein (bovine hemoglobin, BHb) and other proteins (bovine serum albumin, myoglobin, lysozyme and horseradish peroxidase) containing fewer surface-exposed His residues as model proteins...
January 1, 2018: Talanta
https://www.readbyqxmd.com/read/28917052/monitoring-of-methionine-sulfoxide-content-and-methionine-sulfoxide-reductase-activity
#7
Lionel Tarrago, Emmanuel Oheix, Zalán Péterfi, Vadim N Gladyshev
The sulfur-containing amino acid methionine (Met) plays critical roles in protein synthesis, methylation, and sulfur metabolism. Both in its free form and in the form of an amino acid residue, it can be oxidized to the R and S diastereomers of methionine sulfoxide (MetO). Organisms evolved methionine sulfoxide reductases (MSRs) to reduce MetO to Met, with the MSRs type A (MSRA) and type B (MSRB) being specific for the S and R forms of MetO, respectively. In mammals, the selenoprotein MSRB1 plays an important protein repair function, and its expression is tightly regulated by dietary selenium...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28916746/high-yield-production-of-difficult-to-express-proteins-in-a-continuous-exchange-cell-free-system-based-on-cho-cell-lysates
#8
Lena Thoring, Srujan K Dondapati, Marlitt Stech, Doreen A Wüstenhagen, Stefan Kubick
Cell-free protein synthesis (CFPS) represents a promising technology for efficient protein production targeting especially so called "difficult-to-express" proteins whose synthesis is challenging in conventional in vivo protein production platforms. Chinese hamster ovary (CHO) cells are one of the most prominent and safety approved cell lines for industrial protein production. In this study we demonstrated the ability to produce high yields of various protein types including membrane proteins and single chain variable fragments (scFv) in a continuous exchange cell-free (CECF) system based on CHO cell lysate that contains endogenous microsomal structures...
September 15, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28915052/mass-spectrometric-analysis-of-sox11-binding-proteins-in-head-and-neck-cancer-cells-demonstrates-the-interaction-of-sox11-and-hsp90%C3%AE
#9
Naseim Elzakra, Li Cui, Tong Liu, Hong Li, Junwei Huang, Shen Hu
Deregulated expression of SOX11 has been shown to involve in the progression of various types of cancer. However, the role of SOX11 in head and neck cancer remains largely unknown. In this study, co-immunoprecipitation (Co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were performed to identify the proteins that binds to SOX11 at significant higher levels in head and neck cancer cells compared to the normal human oral keratinocytes. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that many potential SOX11-binding partners were associated with protein synthesis, cell metabolism and cell-cell adhesion...
September 15, 2017: Journal of Proteome Research
https://www.readbyqxmd.com/read/28915016/cell-free-protein-synthesis-enhancement-from-real-time-nmr-metabolite-kinetics-redirecting-energy-fluxes-in-hybrid-rrl-systems
#10
Baptiste Panthu, Théophile Ohlmann, Johan Perrier, Uwe Schlattner, Pierre Jalinot, Bénédicte Elena-Herrmann, Gilles J P Rautureau
A counter-intuitive cell-free protein synthesis (CFPS) strategy, based on reducing the ribosomal fraction in rabbit reticulocyte lysate (RRL), triggers the development of hybrid systems composed of RRL ribosome-free supernatant complemented with ribosomes from different mammalian cell-types. Hybrid RRL systems maintain translational properties of the original ribosome cell types, and deliver protein expression levels similar to RRL. Here, we show that persistent ribosome-associated metabolic activity consuming ATP is a major obstacle for maximal protein yield...
September 15, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28892676/a-small-molecule-acts-as-a-roadblock-on-dna-hampering-its-fundamental-processes
#11
Amit Kumar
DNA replication, RNA and protein synthesis are the most fundamental housekeeping processes involved in an organism's growth. Failure or dysregulation of these pathways are often deleterious to life. Therefore, selective inhibition of such processes can be crucial for the inhibition of the growth of any cell, including cancer cells, pathogenic bacteria or other deadly microbes. In the present study, a Zn(2+) complex is shown to act as a roadblock of DNA. The Zn(2+) complex inhibited DNA taq polymerase activity under the in vitro conditions of polymerase chain reaction (PCR)...
September 6, 2017: Journal of Inorganic Biochemistry
https://www.readbyqxmd.com/read/28891200/enhanced-production-of-soluble-recombinant-proteins-with-an-in-situ-removable-fusion-partner-in-a-cell-free-synthesis-system
#12
Devi Kasi, Hee Ju Nah, Christy Catherine, Eung-Soo Kim, Kyubeom Han, Jong-Cheon Ha, Dong-Myung Kim
High-yield production of soluble protein is a common concern in diverse fields of biotechnology. In this study, a strategy of using an engineered nucleotide sequence of ubiquitin for enhancing the production of soluble proteins in a cell-free synthesis system is presented. When examined for a series of proteins that otherwise were poorly expressed, N-terminal fusion with ubiquitin significantly increased both the expression levels and solubility of the translational products. The effect of ubiquitin fusion was also markedly augmented by engineering the nucleotide sequence of ubiquitin, leading to several fold enhancements in soluble production of target proteins...
September 11, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28886561/zinc-protects-against-cadmium-induced-toxicity-by-regulating-oxidative-stress-ions-homeostasis-and-protein-synthesis
#13
Jingmei Pan, Xinhe Huang, Yuxing Li, Ming Li, Ning Yao, Zhengdong Zhou, Xueru Li
The widespread environmental toxin cadmium (Cd) is associated with numerous human diseases. The essential trace element zinc (Zn) strongly counteracts Cd-induced toxicity; however, the mechanism is incompletely understood. Here, we conducted RNA sequencing and bioinformatics analyses to determine the global gene expression profiles of yeast cells exposed to Cd or Cd plus Zn. We identified 912 Cd-induced and 627 Cd plus Zn-induced differentially expressed genes (DEGs). Adding Zn during Cd exposure efficiently reversed the expression of 92...
August 21, 2017: Chemosphere
https://www.readbyqxmd.com/read/28881222/immune-checkpoint-blockade-for-unresectable-or-metastatic-uveal-melanoma-a-systematic-review
#14
REVIEW
Markus V Heppt, Theresa Steeb, Justin Gabriel Schlager, Stefanie Rosumeck, Corinna Dressler, Thomas Ruzicka, Alexander Nast, Carola Berking
BACKGROUND: The use of immune checkpoint blockade (ICB) for uveal melanoma (UM) is little established. The aim of this review was to provide a comprehensive overview on the efficacy, safety, and tolerability of ICB in patients with UM. METHODS: We performed a systematic literature research covering MEDLINE, Embase and CENTRAL. Abstracts of pertinent conferences and trial registers were handsearched for relevant studies. RESULTS: Out of 1327 records initially identified, 12 eligible studies were included in the qualitative synthesis...
August 24, 2017: Cancer Treatment Reviews
https://www.readbyqxmd.com/read/28874504/proteome-analysis-of-hypoxic-glioblastoma-cells-reveals-sequential-metabolic-adaptation-nbsp-of-one-carbon-metabolic-pathways
#15
Kangling Zhang, Pei Xu, James L Sowers, Daniel F Machuca, Barsam Mirfattah, Jason Herring, Hui Tang, Yan Chen, Bing Tian, Allan R Brasier, Lawrence C Sowers
Rapidly proliferating tumors are exposed to a hypoxic microenvironment due to their density, high metabolic consumption, and interruptions in blood flow due to immature angiogenesis. Cellular responses to hypoxia promote highly malignant and metastatic behavior, as well as a chemotherapy-resistant state. In order to better understand the complex relationships between hypoxic adaptations and cancer progression, we studied the dynamic proteome responses of glioblastoma cells exposed to hypoxia via an innovative approach: quantification of newly synthesized proteins using heavy stable-isotope arginine labeling combined with accurate assessment of cell replication by quantification of the light/heavy arginine ratio of peptides in histone H4...
September 5, 2017: Molecular & Cellular Proteomics: MCP
https://www.readbyqxmd.com/read/28867649/synthesis-and-characterization-of-arginine-nipaam-hybrid-hydrogel-as-wound-dressing-in-vitro-and-in-vivo-study
#16
De-Qun Wu, Jie Zhu, Hua Han, Jun-Zhi Zhang, Fei-Fei Wu, Xiao-Hong Qin, Jian-Yong Yu
A multi-functional hybrid hydrogel P(M-Arg/NIPAAm) with temperature response, anti-protein adsorption and antibacterial properties was prepared and applied as wound dressing. The hydrogel was carried out by free radical copolymerization of methacrylate arginine (M-Arg) and N-isopropyl acrylamide (NIPAAm) monomers using N, N'-methylene bisacrylamide as a crosslinker, and ammonium persulfate/N, N, N', N'-tetramethylethylenediamine as the redox initiator. To endow the antimicrobial property, chlorhexidine diacetate (CHX) was preloaded into the hydrogel and polyhexamethylene guanidine phosphate (PHMG) was grafted on the hydrogel surface, respectively...
August 31, 2017: Acta Biomaterialia
https://www.readbyqxmd.com/read/28866808/chylomicrons-stimulate-incretin-secretion-in-mouse-and-human-cells
#17
Arianna Psichas, Pierre F Larraufie, Deborah A Goldspink, Fiona M Gribble, Frank Reimann
AIMS/HYPOTHESIS: Lipids are a potent stimulus for the secretion of glucagon-like peptide (GLP)-1 and glucose-dependent insulinotropic peptide (GIP). Traditionally, this effect was thought to involve the sensing of lipid digestion products by free fatty acid receptor 1 (FFA1) and G-protein coupled receptor 119 (GPR119) on the apical surface of enteroendocrine cells. However, recent evidence suggests that lipids may in fact be sensed basolaterally, and that fatty acid absorption and chylomicron synthesis may be a prerequisite for their stimulatory effect on gut peptide release...
September 2, 2017: Diabetologia
https://www.readbyqxmd.com/read/28864565/a-high-throughput-method-for-the-quantitative-determination-of-free-amino-acids-in-saccharomyces-cerevisiae-by-hydrophilic-interaction-chromatography-tandem-mass-spectrometry
#18
Michael Mülleder, Katharina Bluemlein, Markus Ralser
Amino acids are the building blocks for protein synthesis and the precursors for many biomolecules, such as glutathione and S-adenosylmethionine. Their intracellular concentrations provide valuable information about the overall metabolic state of the cell, as they are closely connected to carbon and nitrogen metabolism and are tightly regulated to meet cellular demands in ever-changing environments. Here, we describe a fast and simple method enabling metabolic profiling for free amino acids for large numbers of yeast strains...
September 1, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28861626/alanyl-glutamine-but-not-glycyl-glutamine-improved-the-proliferation-of-enterocytes-as-glutamine-substitution-in-vitro
#19
Bie Tan, Honglan Liu, Guangzu He, Hao Xiao, Dingfu Xiao, Yanhong Liu, Jianping Wu, Jun Fang, Yulong Yin
The synthetic dipeptides alanyl-glutamine (Ala-Gln) and glycyl-glutamine (Gly-Gln) are used as Gln substitution to provide energy source in the gastrointestinal tract due to their high solubility and stability. This study aimed to investigate the effects of Gln, Ala-Gln and Gly-Gln on mitochondrial respiration and protein turnover of enterocytes. Intestinal porcine epithelial cells (IPEC-J2) were cultured for 2 days in Dulbecco's modified Eagle's-F12 Ham medium (DMEM-F12) containing 2.5 mM Gln, Ala-Gln or Gly-Gln...
August 31, 2017: Amino Acids
https://www.readbyqxmd.com/read/28843001/cell-free-production-of-a-therapeutic-protein-expression-purification-and-characterization-of-recombinant-streptokinase-using-a-cho-lysate
#20
Kevin Tran, Chandrasekhar Gurramkonda, Merideth A Cooper, Manohar Pilli, Joseph Tarris, Nick Selock, Tzu-Chiang Han, Michael Tolosa, Adil Zuber, Chariz Peñalber-Johnstone, Christina Dinkins, Niloufar Pezeshk, Yordan Kostov, Douglas D Frey, Leah Tolosa, David Wood, Govind Rao
The use of cell-free systems to produce recombinant proteins has grown rapidly over the past decade. In particular, cell-free protein synthesis (CFPS) systems based on mammalian cells provide alternative methods for the production of many proteins, including those that contain disulfide bonds, glycosylation and complex structures such as monoclonal antibodies. In the present study, we show robust production of turbo green fluorescent protein (tGFPi and streptokinase in a cell-free system using instrumented mini-bioreactors for highly reproducible protein production...
August 26, 2017: Biotechnology and Bioengineering
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