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https://www.readbyqxmd.com/read/27935292/introduction-of-premature-stop-codons-as-an-evolutionary-strategy-to-rescue-signaling-network-function
#1
Purnima S Kompella, Alan M Moses, Sergio G Peisajovich
The cellular concentrations of key components of signaling networks are tightly regulated, as deviations from their optimal ranges can have negative effects on signaling function. For example, overexpression of the yeast mating pathway mitogen-activated protein kinase (MAPK) Fus3 decreases pathway output, in part by sequestering individual components away from functional multi-protein complexes. Using a synthetic biology approach, we investigated potential mechanisms by which selection could compensate for a decrease in signaling activity caused by overexpression of Fus3...
December 9, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27934767/generation-of-influenza-a-viruses-as-live-but-replication-incompetent-virus-vaccines
#2
Longlong Si, Huan Xu, Xueying Zhou, Ziwei Zhang, Zhenyu Tian, Yan Wang, Yiming Wu, Bo Zhang, Zhenlan Niu, Chuanling Zhang, Ge Fu, Sulong Xiao, Qing Xia, Lihe Zhang, Demin Zhou
The conversion of life-threatening viruses into live but avirulent vaccines represents a revolution in vaccinology. In a proof-of-principle study, we expanded the genetic code of the genome of influenza A virus via a transgenic cell line containing orthogonal translation machinery. This generated premature termination codon (PTC)-harboring viruses that exerted full infectivity but were replication-incompetent in conventional cells. Genome-wide optimization of the sites for incorporation of multiple PTCs resulted in highly reproductive and genetically stable progeny viruses in transgenic cells...
December 2, 2016: Science
https://www.readbyqxmd.com/read/27924918/production-of-phloroglucinol-a-platform-chemical-in-arabidopsis-using-a-bacterial-gene
#3
Salah E Abdel-Ghany, Irene Day, Adam L Heuberger, Corey D Broeckling, Anireddy S N Reddy
Phloroglucinol (1,3,5-trihydroxybenzene; PG) and its derivatives are phenolic compounds that are used for various industrial applications. Current methods to synthesize PG are not sustainable due to the requirement for carbon-based precursors and co-production of toxic byproducts. Here, we describe a more sustainable production of PG using plants expressing a native bacterial or a codon-optimized synthetic PhlD targeted to either the cytosol or chloroplasts. Transgenic lines were analyzed for the production of PG using gas and liquid chromatography coupled to mass spectroscopy...
December 7, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27915289/codon-usage-is-less-optimized-in-eukaryotic-gene-segments-encoding-intrinsically-disordered-regions-than-in-those-encoding-structural-domains
#4
Keiichi Homma, Tamotsu Noguchi, Satoshi Fukuchi
Codon usage tends to be optimized in highly expressed genes. A plausible explanation for this phenomenon is that translational accuracy is increased in highly expressed genes with infrequent use of rare codons. Besides structural domains (SDs), eukaryotic proteins generally have intrinsically disordered regions (IDRs) that by themselves do not assume unique three-dimensional structures. As IDRs are free from structural constraint, they can probably accommodate more translational errors than SDs can. Thus, codon usage in IDRs is likely to be less optimized than that in SDs...
December 1, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27912985/immunogenicity-and-malaria-transmission-reducing-potency-of-pfs48-45-and-pfs25-encoded-by-dna-vaccines-administered-by-intramuscular-electroporation
#5
Dibyadyuti Datta, Geetha P Bansal, Dietlind L Gerloff, Barry Ellefsen, Drew Hannaman, Nirbhay Kumar
Pfs48/45 and Pfs25 are leading candidates for the development of Plasmodium falciparum transmission blocking vaccines (TBV). Expression of Pfs48/45 in the erythrocytic sexual stages and presentation to the immune system during infection in the human host also makes it ideal for natural boosting. However, it has been challenging to produce a fully folded, functionally active Pfs48/45, using various protein expression platforms. In this study, we demonstrate that full-length Pfs48/45 encoded by DNA plasmids is able to induce significant transmission reducing immune responses...
November 29, 2016: Vaccine
https://www.readbyqxmd.com/read/27905914/development-of-a-gene-synthesis-platform-for-the-efficient-large-scale-production-of-small-genes-encoding-animal-toxins
#6
Ana Filipa Sequeira, Joana L A Brás, Catarina I P D Guerreiro, Renaud Vincentelli, Carlos M G A Fontes
BACKGROUND: Gene synthesis is becoming an important tool in many fields of recombinant DNA technology, including recombinant protein production. De novo gene synthesis is quickly replacing the classical cloning and mutagenesis procedures and allows generating nucleic acids for which no template is available. In addition, when coupled with efficient gene design algorithms that optimize codon usage, it leads to high levels of recombinant protein expression. RESULTS: Here, we describe the development of an optimized gene synthesis platform that was applied to the large scale production of small genes encoding venom peptides...
December 1, 2016: BMC Biotechnology
https://www.readbyqxmd.com/read/27903094/effects-of-self-complementarity-codon-optimization-transgene-and-dose-on-liver-transduction-with-aav8
#7
Peter Bell, Lili Wang, Shu-Jen Chen, Hongwei Yu, Yanqing Zhu, Mohamad Nayal, Zhenning He, John White, Deborah Lebel-Hagan, James M Wilson
Numerous methods of vector design and delivery have been employed in an attempt to increase transgene expression following AAV-based gene therapy. Here, we conducted a gene transfer study in mice to compare the effects of vector self-complementarity (double- or single-stranded DNA), codon optimization of the transgene, and vector dose on transgene expression levels in liver. Two different reporter genes were used: human ornithine transcarbamylase (hOTC) detected by immunofluorescence, and enhanced green fluorescent protein (EGFP) detected by direct fluorescence...
November 30, 2016: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/27897254/high-level-expression-of-improved-thermo-stable-alkaline-xylanase-variant-in-pichia-pastoris-through-codon-optimization-multiple-gene-insertion-and-high-density-fermentation
#8
Yihong Lu, Cheng Fang, Qinhong Wang, Yuling Zhou, Guimin Zhang, Yanhe Ma
In paper industry, xylanases are used to increase the pulp properties in bleaching process as its eco-friendly nature. The xylanases activity is hindered by high temperature and alkaline conditions with high enzyme production cost in the paper industry. Here, XynHB, an alkaline stable xylanase from Bacillus pumilus HBP8 was mutated at N188A to XynHBN188A. Expressed mutant in E. coli showed 1.5-fold higher xylanase activity than XynHB at 60 °C. The mutant expressed in Pichia pastoris was glycosylated, remained stable for 30 min at 60 °C...
November 29, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27896426/expression-and-characterization-of-a-codon-optimized-alkaline-stable-carbonic-anhydrase-from-aliivibrio-salmonicida-for-co2-sequestration-applications
#9
So-Young Jun, Sung Ho Kim, Bashista Kumar Kanth, Jinwon Lee, Seung Pil Pack
The CO2 mineralization process, accelerated by carbonic anhydrase (CA) was proposed for the efficient capture and storage of CO2, the accumulation of which in the atmosphere is the main cause of global warming. Here, we characterize a highly stable form of the cloned CA from the Gram-negative marine bacterium Aliivibrio salmonicida, named ASCA that can promote CO2 absorption in an alkaline solvent required for efficient carbon capture. We designed a mature form of ASCA (mASCA) using a codon optimization of ASCA gene and removal of ASCA signal peptide...
November 28, 2016: Bioprocess and Biosystems Engineering
https://www.readbyqxmd.com/read/27893966/vaccination-via-chloroplast-genetics-affordable-protein-drugs-for-the-prevention-and-treatment-of-inherited-or-infectious-human-diseases
#10
Henry Daniell, Hui-Ting Chan, Elise K Pasoreck
Plastid-made biopharmaceuticals treat major metabolic or genetic disorders, including Alzheimer's, diabetes, hypertension, hemophilia, and retinopathy. Booster vaccines made in chloroplasts prevent global infectious diseases, such as tuberculosis, malaria, cholera, and polio, and biological threats, such as anthrax and plague. Recent advances in this field include commercial-scale production of human therapeutic proteins in FDA-approved cGMP facilities, development of tags to deliver protein drugs to targeted human cells or tissues, methods to deliver precise doses, and long-term stability of protein drugs at ambient temperature, maintaining their efficacy...
November 23, 2016: Annual Review of Genetics
https://www.readbyqxmd.com/read/27893794/single-amino-acid-repeats-in-the-proteome-world-structural-functional-and-evolutionary-insights
#11
Amitha Sampath Kumar, Divya Tej Sowpati, Rakesh K Mishra
Microsatellites or simple sequence repeats (SSR) are abundant, highly diverse stretches of short DNA repeats present in all genomes. Tandem mono/tri/hexanucleotide repeats in the coding regions contribute to single amino acids repeats (SAARs) in the proteome. While SSRs in the coding region always result in amino acid repeats, a majority of SAARs arise due to a combination of various codons representing the same amino acid and not as a consequence of SSR events. Certain amino acids are abundant in repeat regions indicating a positive selection pressure behind the accumulation of SAARs...
2016: PloS One
https://www.readbyqxmd.com/read/27887869/a-highly-diverse-and-functional-na%C3%A3-ve-ubiquitin-variant-library-for-generation-of-intracellular-affinity-reagents
#12
Isabel Leung, Nick Jarvik, Sachdev S Sidhu
We report the design, construction, and validation of a highly diverse phage-displayed naïve ubiquitin variant (Ubv) library. We first conducted a mutation tolerance scan of 27 residues and confirmed that 24 of these could be substituted by chemically diverse amino acids without compromising the display of Ubvs on phage. Subsequently, we constructed a library containing 6.8×10(10) unique members, in which these 24 positions were diversified with a degenerate codon that encodes for 6 aa that are prevalent in protein interaction sites...
November 22, 2016: Journal of Molecular Biology
https://www.readbyqxmd.com/read/27881683/rational-design-of-glycoengineered-interferon-%C3%AE-analogs-with-improved-aggregation-state-experimental-validation
#13
M Samoudi, Z Minuchehr, S W Harcum, F Tabandeh, N Omid Yeganeh, M Khodabandeh
Recombinant human interferon-β (rhIFN-β) used clinically has lower efficacy than expected due to protein instabilities such as aggregation. Increasing molecular stability, glycoengineering has been used to improve clinical efficacy for a number of therapeutics; however, often labor-intensive trail-and-error approaches are used to identify additional glycosylation sites. In this study two rhIFN-β analogs with one additional glycosylation site, L6T and S75N, identified by a rational in silico approach, were characterized...
November 23, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27872686/engineering-translation-in-mammalian-cell-factories-to-increase-protein-yield-the-unexpected-use-of-long-non-coding-sineup-rnas
#14
REVIEW
Silvia Zucchelli, Laura Patrucco, Francesca Persichetti, Stefano Gustincich, Diego Cotella
Mammalian cells are an indispensable tool for the production of recombinant proteins in contexts where function depends on post-translational modifications. Among them, Chinese Hamster Ovary (CHO) cells are the primary factories for the production of therapeutic proteins, including monoclonal antibodies (MAbs). To improve expression and stability, several methodologies have been adopted, including methods based on media formulation, selective pressure and cell- or vector engineering. This review presents current approaches aimed at improving mammalian cell factories that are based on the enhancement of translation...
2016: Computational and Structural Biotechnology Journal
https://www.readbyqxmd.com/read/27866333/strain-engineering-and-process-optimization-for-enhancing-the-production-of-a-thermostable-steryl-glucosidase-in-escherichia-coli
#15
Florencia Eberhardt, Andres Aguirre, Hugo G Menzella, Salvador Peiru
Biodiesels produced from transesterification of vegetable oils have a major problem in quality due to the presence of precipitates, which are mostly composed of steryl glucosides (SGs). We have recently described an enzymatic method for the efficient removal of SGs from biodiesel, based on the activity of a thermostable β-glycosidase from Thermococcus litoralis. In the present work, we describe the development of an Escherichia coli-based expression system and a high cell density fermentation process. Strain and process engineering include the assessment of different promoters to drive the expression of a codon-optimized gene, the co-expression of molecular chaperones and the development of a high cell density fermentation process...
November 19, 2016: Journal of Industrial Microbiology & Biotechnology
https://www.readbyqxmd.com/read/27866312/transgenic-citrus-expressing-synthesized-cecropin-b-genes-in-the-phloem-exhibits-decreased-susceptibility-to-huanglongbing
#16
Xiuping Zou, Xueyou Jiang, Lanzhen Xu, Tiangang Lei, Aihong Peng, Yongrui He, Lixiao Yao, Shanchun Chen
Expression of synthesized cecropin B genes in the citrus phloem, where Candidatus Liberibacter asiaticus resides, significantly decreased host susceptibility to Huanglongbing. Huanglongbing (HLB), associated with Candidatus Liberibacter asiaticus bacteria, is the most destructive disease of citrus worldwide. All of the commercial sweet orange cultivars lack resistance to this disease. The cationic lytic peptide cecropin B, isolated from the Chinese tasar moth (Antheraea pernyi), has been shown to effectively eliminate bacteria...
November 19, 2016: Plant Molecular Biology
https://www.readbyqxmd.com/read/27864159/high-level-expression-and-efficient-refolding-of-therapeutically-important-recombinant-human-interleukin-3-hil-3-in-e-%C3%A2-coli
#17
Vikas Kumar Dagar, Adivitiya, Yogender Pal Khasa
Human interleukin-3 (hIL-3) is a pleiotropic cytokine that stimulates the differentiation and proliferation of multipotent hematopoietic cells thus making it a therapeutically important molecule. In this study, its poor expression yield was improved by addressing various upstream bottlenecks in E. coli heterologous system. The codon-optimized hIL-3 gene was cloned under various signal sequences and solubility enhancer fusion tags for its hyper-expression under a strong T7 promoter. The optimization of shake flask expression studies resulted in a hIL-3 protein concentration of 225 mg/L in the form of inclusion bodies (IBs)...
November 15, 2016: Protein Expression and Purification
https://www.readbyqxmd.com/read/27842546/optimization-of-construct-design-and-fermentation-strategy-for-the-production-of-bioactive-atf-sap-a-saporin-based-anti-tumoral-upar-targeted-chimera
#18
Alfredo Errico Provenzano, Riccardo Posteri, Francesco Giansanti, Francesco Angelucci, Sopsamorn U Flavell, David J Flavell, Maria Serena Fabbrini, Danilo Porro, Rodolfo Ippoliti, Aldo Ceriotti, Paola Branduardi, Riccardo Vago
BACKGROUND: The big challenge in any anti-tumor therapeutic approach is represented by the development of drugs selectively acting on the target with limited side effects, that exploit the unique characteristics of malignant cells. The urokinase (urokinase-type plasminogen activator, uPA) and its receptor uPAR have been identified as preferential target candidates since they play a key role in the evolution of neoplasms and are associated with neoplasm aggressiveness and poor clinical outcome in several different tumor types...
November 14, 2016: Microbial Cell Factories
https://www.readbyqxmd.com/read/27842439/stable-in-vivo-transgene-expression-in-endothelial-cells-with-helper-dependent-adenovirus-roles-of-promoter-and-interleukin-10
#19
Nagadhara Dronadula, Bradley Wacker, Reginald Van Der Kwast, Jingwan Zhang, David A Dichek
Our long-term goal is to prevent or reverse atherosclerosis by delivering gene therapy from stably transduced endothelial cells (EC). We previously reported that EC-directed gene therapy with a helper-dependent adenovirus (HDAd) expressing apolipoprotein A-I (apo A-I) retarded development of atherosclerosis in rabbit carotid arteries over a 1-month interval. However, a 70% decline in apo A-I expression during this time raised concerns about long-term efficacy of this approach. Here we report use of several approaches aimed either at preventing this decline or at increasing apo A-I expression from HDAd at all time points: codon optimization, deletion of 3´ untranslated sequences, substitution of a synthetic mammalian-based promoter (4XETE) for the CMV promoter, and co-transduction with an HDAd expressing interleukin-10...
November 14, 2016: Human Gene Therapy
https://www.readbyqxmd.com/read/27826796/bioengineering-of-the-plant-culture-of-capsicum-frutescens-with-vanillin-synthase-gene-for-the-production-of-vanillin
#20
Marcus Jenn Yang Chee, Grantley W Lycett, Teng-Jin Khoo, Chiew Foan Chin
Production of vanillin by bioengineering has gained popularity due to consumer demand toward vanillin produced by biological systems. Natural vanillin from vanilla beans is very expensive to produce compared to its synthetic counterpart. Current bioengineering works mainly involve microbial biotechnology. Therefore, alternative means to the current approaches are constantly being explored. This work describes the use of vanillin synthase (VpVAN), to bioconvert ferulic acid to vanillin in a plant system. The VpVAN enzyme had been shown to directly convert ferulic acid and its glucoside into vanillin and its glucoside, respectively...
November 8, 2016: Molecular Biotechnology
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