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Heterologous expression lab

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https://www.readbyqxmd.com/read/28331545/metabolic-engineering-of-saccharomyces-cerevisiae-to-produce-a-reduced-viscosity-oil-from-lignocellulose
#1
Tam N T Tran, Rebecca J Breuer, Ragothaman Avanasi Narasimhan, Lucas S Parreiras, Yaoping Zhang, Trey K Sato, Timothy P Durrett
BACKGROUND: Acetyl-triacylglycerols (acetyl-TAGs) are unusual triacylglycerol (TAG) molecules that contain an sn-3 acetate group. Compared to typical triacylglycerol molecules (here referred to as long chain TAGs; lcTAGs), acetyl-TAGs possess reduced viscosity and improved cold temperature properties, which may allow direct use as a drop-in diesel fuel. Their different chemical and physical properties also make acetyl-TAGs useful for other applications such as lubricants and plasticizers...
2017: Biotechnology for Biofuels
https://www.readbyqxmd.com/read/28321412/recombinant-lactococcus-lactis-expressing-haemagglutinin-from-a-polish-avian-h5n1-isolate-and-its-immunological-effect-in-preliminary-animal-trials
#2
Agnieszka K Szczepankowska, Katarzyna Szatraj, Przemysław Sałański, Agnieszka Rózga, Roman K Górecki, Jacek K Bardowski
Lactic acid bacteria (LAB) are Gram-positive, nonpathogenic microorganisms that are gaining much interest as antigen producers for development of live vaccine vectors. Heterologous proteins of different origin have been successfully expressed in various LAB species, including Lactococcus lactis. Recombinant L. lactis strains have been shown to induce specific local and systemic immune responses against various antigens. Our study aimed at constructing a L. lactis strain expressing haemagglutinin of a Polish avian H5H1 influenza isolate and examining its effect on animals...
2017: BioMed Research International
https://www.readbyqxmd.com/read/28315210/recombinant-allergens-production-in-yeast
#3
Maria Neophytou, Marcos Alcocer
The methylotropic yeast Pichia pastoris has been extensively used in large-scale production of properly folded recombinant proteins. As an eukaryotic organism P. pastoris presents a series of advantages at expression and processing of heterologous proteins such as post-translational modifications, protein processing, and a reasonably sophisticated quality control of protein folding when compared against Escherichia coli. In this chapter, we describe the modified lab procedure for cloning and expression in Pichia pastoris of common food allergens sequences from the raw fruit to the fully folded biotinylated protein product...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28288336/breaking-the-silence-new-strategies-for-discovering-novel-natural-products
#4
REVIEW
Hengqian Ren, Bin Wang, Huimin Zhao
Natural products have been a prolific source of antibacterial and anticancer drugs for decades. One of the major challenges in natural product discovery is that the vast majority of natural product biosynthetic gene clusters (BGCs) have not been characterized, partially due to the fact that they are either transcriptionally silent or expressed at very low levels under standard laboratory conditions. Here we describe the strategies developed in recent years (mostly between 2014-2016) for activating silent BGCs...
March 10, 2017: Current Opinion in Biotechnology
https://www.readbyqxmd.com/read/28265993/real-time-genetic-manipulations-of-the-cytokinin-pathway-a-tool-for-laboratory-and-field-studies
#5
Martin Schäfer, Stefan Meldau
Although many established tools for cytokinin (CK) pathway manipulations are well suitable for the analysis of molecular interactions, their use on a whole plant scale is often limited by the induction of severe developmental defects. To circumvent this problem, different methods were developed that allow for a more precise manipulation of the CK pathway. Here we present one of these systems, the pOp6/LhGR system for chemically inducible gene expression. This system allows regulation on a spatial, temporal, and quantitative scale and therefore provides a superior tool for analyzing the role of CKs in the interactions of plants with their environment...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28210796/optogenetic-switches-for-light-controlled-gene-expression-in-yeast
#6
REVIEW
Francisco Salinas, Vicente Rojas, Verónica Delgado, Eduardo Agosin, Luis F Larrondo
Light is increasingly recognized as an efficient means of controlling diverse biological processes with high spatiotemporal resolution. Optogenetic switches are molecular devices for regulating light-controlled gene expression, protein localization, signal transduction and protein-protein interactions. Such molecular components have been mainly developed through the use of photoreceptors, which upon light stimulation undergo conformational changes passing to an active state. The current repertoires of optogenetic switches include red, blue and UV-B light photoreceptors and have been implemented in a broad spectrum of biological platforms...
February 16, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28117773/xenopus-oocytes-optimized-methods-for-microinjection-removal-of-follicular-cell-layers-and-fast-solution-changes-in-electrophysiological-experiments
#7
Maria C Maldifassi, Nisa Wongsamitkul, Roland Baur, Erwin Sigel
The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.(1) and has been widely used since its discovery (References 2 - 3, and references therein). A characteristic that makes the oocyte attractive for foreign channel expression is the poor abundance of endogenous ion channels(4). This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels. The expression of GABAA receptors in Xenopus oocytes and their functional characterization is described here, including the isolation of oocytes, microinjections with cRNA, the removal of follicular cell layers, and fast solution changes in electrophysiological experiments...
December 31, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28075554/an-engineered-allele-of-afsq1-facilitates-the-discovery-and-investigation-of-cryptic-natural-products
#8
Martin Daniel-Ivad, Nabeela Hameed, Stephanie Tan, Rachna Dhanjal, Daniel Socko, Patricia Pak, Tomas Gverzdys, Marie A Elliot, Justin R Nodwell
New approaches to antimicrobial discovery are needed to address the growing threat of antibiotic resistance. The Streptomyces genus, a proven source of antibiotics, is recognized as having a large reservoir of untapped secondary metabolic genes, many of which are likely to produce uncharacterized compounds. However, most of these compounds are currently inaccessible, as they are not expressed under standard laboratory conditions. Here, we present a novel methodology for activating these "cryptic" metabolites by heterologously expressing a constitutively active pleiotropic regulator...
January 19, 2017: ACS Chemical Biology
https://www.readbyqxmd.com/read/27981234/coordinate-regulation-of-antimycin-and-candicidin-biosynthesis
#9
Thomas C McLean, Paul A Hoskisson, Ryan F Seipke
Streptomyces species produce an incredible array of high-value specialty chemicals and medicinal therapeutics. A single species typically harbors ~30 biosynthetic pathways, but only a few them are expressed in the laboratory; thus, poor understanding of how natural-product biosynthesis is regulated is a major bottleneck in drug discovery. Antimycins are a large family of anticancer compounds widely produced by Streptomyces species, and their regulation is atypical compared to that of most other natural products...
November 2016: MSphere
https://www.readbyqxmd.com/read/27906033/the-role-of-the-backbone-torsion-in-protein-folding
#10
EDITORIAL
Irina Sorokina, Arcady Mushegian
BACKGROUND: The set of forces and sequence of events that govern the transition from an unfolded polypeptide chain to a functional protein with correct spatial structure remain incompletely known, despite the importance of the problem and decades of theory development, computer simulations, and laboratory experiments. Information about the correctly folded state of most proteins is likely to be present in their sequences, and yet many proteins fail to attain native structure after overexpression in a non-native environment or upon experimental denaturation and refolding...
December 1, 2016: Biology Direct
https://www.readbyqxmd.com/read/27888410/genome-sequence-prevalence-and-quantification-of-the-first-iflavirus-identified-in-a-phytoplasma-insect-vector
#11
Simona Abbà, Luciana Galetto, Marta Vallino, Marika Rossi, Massimo Turina, Anne Sicard, Cristina Marzachì
The leafhopper Euscelidius variegatus is a natural vector of chrysanthemum yellows phytoplasma (CY) and an efficient vector of flavescence dorée phytoplasma (FD) under laboratory conditions. During a transcriptome sequencing (RNA-seq) project aimed at investigating the interactions between the insect and the two phytoplasmas, a 10,616-nucleotide-long contig with high sequence similarity to known picorna-like viruses was identified among the assembled insect transcripts. The discovery came totally unexpected, because insects from the laboratory colony did not show any evident symptom that could be related to the presence of a virus...
March 2017: Archives of Virology
https://www.readbyqxmd.com/read/27830495/cloning-a-novel-endo-1-4-%C3%AE-d-glucanase-gene-from-trichoderma-virens-and-heterologous-expression-in-e-coli
#12
Rong Zeng, Qiao Hu, Xiao-Yan Yin, Hao Huang, Jia-Bao Yan, Zhi-Wei Gong, Zhong-Hua Yang
Endo-1,4-β-D-glucanase (EG), as a key constituent of cellulase taking the responsibility of cutting β-1,4 glycosidic bonds, plays the essential role in the process of degrading cellulose by cellulase. Cloning and expressing the EG gene is important to the cellulase research and application. In this work, a novel EG gene was cloned from Trichoderma virens ZY-01, which was a cellulase secreting microbe isolated by our laboratory. The DNA sequence showed that the length of the cloned EG is 1069 bp, which had 95...
December 2016: AMB Express
https://www.readbyqxmd.com/read/27816999/heterologous-expression-of-the-crassostrea-gigas-pacific-oyster-alternative-oxidase-in-the-yeast-saccharomyces-cerevisiae
#13
Aaron Robertson, Kyle Schaltz, Karina Neimanis, James F Staples, Allison E McDonald
Alternative oxidase (AOX) is a terminal oxidase within the inner mitochondrial membrane (IMM) present in many organisms where it functions in the electron transport system (ETS). AOX directly accepts electrons from ubiquinol and is therefore capable of bypassing ETS Complexes III and IV. The human genome does not contain a gene coding for AOX, so AOX expression has been suggested as a gene therapy for a range of human mitochondrial diseases caused by genetic mutations that render Complex III and/or IV dysfunctional...
October 2016: Journal of Bioenergetics and Biomembranes
https://www.readbyqxmd.com/read/27745812/meningococcal-serogroup-b-specific-responses-after-vaccination-with-bivalent-rlp2086-4-year-follow-up-of-a-randomised-single-blind-placebo-controlled-phase-2-trial
#14
Helen S Marshall, Peter C Richmond, Johannes Beeslaar, Qin Jiang, Kathrin U Jansen, Maria Garcés-Sánchez, Federico Martinón-Torres, Leszek Szenborn, Jacek Wysocki, Joseph Eiden, Shannon L Harris, Thomas R Jones, Su-San Lee, John L Perez
BACKGROUND: Bivalent rLP2086 is a recombinant factor H binding protein-based vaccine approved in the USA for prevention of meningococcal serogroup B disease in 10-25-year-olds. We aimed to assess the persistence of bactericidal antibodies up to 4 years after a three-dose schedule of bivalent rLP2086. METHODS: We did this randomised, single-blind, placebo-controlled, phase 2 trial at 25 sites in Australia, Poland, and Spain. In stage 1 of the study (February, 2009-May, 2010), healthy adolescents (aged 11-18 years) were randomly assigned, via an interactive voice and web-response system with computer-generated sequential random numbers, to receive either ascending doses of vaccine (60 μg, 120 μg, and 200 μg) or placebo at months 0, 2, and 6...
January 2017: Lancet Infectious Diseases
https://www.readbyqxmd.com/read/27692771/identification-of-plasmodium-falciparum-reticulocyte-binding-protein-homologue-5-interacting-protein-pfripr-as-a-highly-conserved-blood-stage-malaria-vaccine-candidate
#15
Edward H Ntege, Nobuko Arisue, Daisuke Ito, Tomoyuki Hasegawa, Nirianne M Q Palacpac, Thomas G Egwang, Toshihiro Horii, Eizo Takashima, Takafumi Tsuboi
Genetic variability in Plasmodium falciparum malaria parasites hampers current malaria vaccine development efforts. Here, we hypothesize that to address the impact of genetic variability on vaccine efficacy in clinical trials, conserved antigen targets should be selected to achieve robust host immunity across multiple falciparum strains. Therefore, suitable vaccine antigens should be assessed for levels of polymorphism and genetic diversity. Using a total of one hundred and two clinical isolates from a region of high malaria transmission in Uganda, we analyzed extent of polymorphism and genetic diversity in four recently reported novel blood-stage malaria vaccine candidate proteins: Rh5 interacting protein (PfRipr), GPI anchored micronemal antigen (PfGAMA), rhoptry-associated leucine zipper-like protein 1 (PfRALP1) and Duffy binding-like merozoite surface protein 1 (PfMSPDBL1)...
September 28, 2016: Vaccine
https://www.readbyqxmd.com/read/27609229/secretion-of-m2e-hbc-fusion-protein-by-lactobacillus-casei-using-cwh-signal-peptide
#16
Sirintra Themsakul, Namfon Suebwongsa, Baltasar Mayo, Marutpong Panya, Viraphong Lulitanond
The ability to serve as a delivery vehicle for various interesting biomolecules makes lactic acid bacteria (LAB) very useful in several applications. In the medical field, recombinant LAB expressing pathogenic antigens at different cellular locations have been used to elicit both mucosal and systemic immune responses. Expression-secretion vectors (ESVs) with a signal peptide (SP) are pivotal for protein expression and secretion. In this study, the genome sequence of Lactobacillus casei ATCC334 was explored for new SPs using bioinformatics tools...
October 2016: FEMS Microbiology Letters
https://www.readbyqxmd.com/read/27538995/roseolovirus-associated-encephalitis-in-immunocompetent-and-immunocompromised-individuals
#17
REVIEW
Joseph Ongrádi, Dharam V Ablashi, Tetsushi Yoshikawa, Balázs Stercz, Masao Ogata
The roseoloviruses, human herpesvirus (HHV)-6A, HHV-6B, and HHV-7, can cause severe encephalitis or encephalopathy. In immunocompetent children, primary HHV-6B infection is occasionally accompanied by diverse clinical forms of encephalitis. Roseolovirus coinfections with heterologous viruses and delayed primary HHV-7 infection in immunocompetent adults result in very severe neurological and generalized symptoms. Recovery from neurological sequelae is slow and sometimes incomplete. In immunocompromised patients with underlying hematological malignancies and transplantation, frequent single or simultaneous reactivation of roseoloviruses elicit severe, lethal organ dysfunctions, including damages in the limbic system, brain stem, and hippocampus...
February 2017: Journal of Neurovirology
https://www.readbyqxmd.com/read/27486448/transfer-of-a-catabolic-pathway-for-chloromethane-in-methylobacterium-strains-highlights-different-limitations-for-growth-with-chloromethane-or-with-dichloromethane
#18
Joshua K Michener, Stéphane Vuilleumier, Françoise Bringel, Christopher J Marx
Chloromethane (CM) is an ozone-depleting gas, produced predominantly from natural sources, that provides an important carbon source for microbes capable of consuming it. CM catabolism has been difficult to study owing to the challenging genetics of its native microbial hosts. Since the pathways for CM catabolism show evidence of horizontal gene transfer, we reproduced this transfer process in the laboratory to generate new CM-catabolizing strains in tractable hosts. We demonstrate that six putative accessory genes improve CM catabolism, though heterologous expression of only one of the six is strictly necessary for growth on CM...
2016: Frontiers in Microbiology
https://www.readbyqxmd.com/read/27382786/-progress-in-heterologous-expression-of-fungal-natural-products-a-review
#19
REVIEW
Zihui Ma, Wei Li, Wenbing Yin
Fungal products are important resources for natural drugs. Availabilities of many fungal genome sequences indicate that fungi have the huge potential to produce secondary metabolites. However, many valuable fungal natural products are hard to obtain under regular lab conditions because fungi are slow to grow, difficult to genetically modify, or have low yield of desired metabolites. Therefore, developing a heterologous expression system has been an efficient approach to discover novel natural product and elucidate the complicated biosynthetic pathways...
March 4, 2016: Wei Sheng Wu Xue Bao, Acta Microbiologica Sinica
https://www.readbyqxmd.com/read/27381522/insights-into-microbial-cryptic-gene-activation-and-strain-improvement-principle-application-and-technical-aspects
#20
Kozo Ochi
As bacteria and fungi have been found to contain genes encoding enzymes that synthesize a plethora of potential secondary metabolites, interest has grown in the activation of these cryptic pathways. Homologous and heterologous expression of these cryptic secondary metabolite-biosynthetic genes, often silent under ordinary laboratory fermentation conditions, may lead to the discovery of novel secondary metabolites. This review addresses current progress in the activation of these pathways, describing methods for activating silent genes...
July 6, 2016: Journal of Antibiotics
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