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https://www.readbyqxmd.com/read/28935113/directed-evolution-of-glycopeptides-using-mrna-display
#1
Satoru Horiya, Jennifer K Bailey, Isaac J Krauss
Directed evolution is a useful method for the discovery of nucleic acids, peptides, or proteins that have desired binding abilities or functions. Because of the abundance and importance of glycosylation in nature, directed evolution of glycopeptides and glycoproteins is also highly desirable. However, common directed evolution platforms such as phage-, yeast-, or mammalian-cell display are limited for these applications by several factors. Glycan structure at each glycosylation site is not genetically encoded, and yeast and mammalian cells produce a heterogeneous mixture of glycoforms at each site on the protein...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28928140/selenoprotein-t-is-a-novel-ost-subunit-that-regulates-upr-signaling-and-hormone-secretion
#2
Abdallah Hamieh, Dorthe Cartier, Houssni Abid, André Calas, Carole Burel, Christine Bucharles, Cedric Jehan, Luca Grumolato, Marc Landry, Patrice Lerouge, Youssef Anouar, Isabelle Lihrmann
Selenoprotein T (SelT) is a recently characterized thioredoxin-like protein whose expression is very high during development, but is confined to endocrine tissues in adulthood where its function is unknown. We report here that SelT is required for adaptation to the stressful conditions of high hormone level production in endocrine cells. Using immunofluorescence and TEM immunogold approaches, we find that SelT is expressed at the endoplasmic reticulum membrane in all hormone-producing pituitary cell types. SelT knockdown in corticotrope cells promotes unfolded protein response (UPR) and ER stress and lowers endoplasmic reticulum-associated protein degradation (ERAD) and hormone production...
September 19, 2017: EMBO Reports
https://www.readbyqxmd.com/read/28924963/combining-metabolic-and-process-engineering-strategies-to-improve-recombinant-glycoprotein-production-and-quality
#3
Eric Karengera, Yves Durocher, Gregory De Crescenzo, Olivier Henry
Increasing recombinant protein production while ensuring a high and consistent protein quality remains a challenge in mammalian cell culture process development. In this work, we combined a nutrient substitution approach with a metabolic engineering strategy that improves glucose utilization efficiency. This combination allowed us to tackle both lactate and ammonia accumulation and investigate on potential synergistic effects on protein production and quality. To this end, HEK293 cells overexpressing the pyruvate yeast carboxylase (PYC2) and their parental cells, both stably producing the therapeutic glycoprotein interferon α2b (IFNα2b), were cultured in media deprived of glutamine but containing chosen substitutes...
September 18, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28921426/production-of-full-length-antibody-by-pichia-pastoris
#4
Adam Nylen, Ming-Tang Chen
The methylotrophic yeast Pichia pastoris has become an increasingly popular host for recombinant protein expression in recent times. MRL pioneered a glycoengineered humanized P. pastoris expression system that could produce glycoproteins with glycosylation profiles similar to mammalian systems. Therapeutic glycoproteins produced by the humanized P. pastoris platform have shown comparable folding, stability, and in vitro and in vivo efficacies in preclinical models to their counterparts produced from the CHO cells...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28857681/expression-and-purification-of-the-extracellular-domains-of-human-glycoprotein-vi-gp-vi-and-the-receptor-for-advanced-glycation-end-products-rage-from-rattus-norvegicus-in-leishmania-tarentolae
#5
Thomas Langer, Carsten Corvey, Katja Kroll, Oliver Boscheinen, Thomas Wendrich, Werner Dittrich
Glycosylation is one of the most complex posttranslational modifications and may have significant influence on the proper function of the corresponding proteins. Bacteria and yeast are, because of easy handling and cost reasons, the most frequently used systems for recombinant protein expression. Bacteria generally do not glycosylate proteins and yeast might tend to hyperglycosylate. Insect cell and mammalian cell based expression system are able to produce complex N-glycosylation structures, but are more complex to handle and more expensive...
August 31, 2017: Preparative Biochemistry & Biotechnology
https://www.readbyqxmd.com/read/28810660/recent-advancements-in-understanding-mammalian-o-mannosylation
#6
M Osman Sheikh, Stephanie M Halmo, Lance Wells
The post-translational glycosylation of select proteins by O-linked mannose (O-mannose or O-man) is a conserved modification from yeast to humans and has been shown to be necessary for proper development and growth. The most well studied O-mannosylated mammalian protein is α-dystroglycan (α-DG). Hypoglycosylation of α-DG results in varying severities of congenital muscular dystrophies, cancer progression and metastasis, and inhibited entry and infection of certain arenaviruses. Defects in the gene products responsible for post-translational modification of α-DG, primarily glycosyltransferases, are the basis for these diseases...
September 1, 2017: Glycobiology
https://www.readbyqxmd.com/read/28797224/the-adaptive-landscape-of-wildtype-and-glycosylation-deficient-populations-of-the-industrial-yeast-pichia-pastoris
#7
Josef W Moser, Iain B H Wilson, Martin Dragosits
BACKGROUND: The effects of long-term environmental adaptation and the implications of major cellular malfunctions are still poorly understood for non-model but biotechnologically relevant species. In this study we performed a large-scale laboratory evolution experiment with 48 populations of the yeast Pichia pastoris in order to establish a general adaptive landscape upon long-term selection in several glucose-based growth environments. As a model for a cellular malfunction the implications of OCH1 mannosyltransferase knockout-mediated glycosylation-deficiency were analyzed...
August 10, 2017: BMC Genomics
https://www.readbyqxmd.com/read/28794948/glucosomes-glycosylated-vesicle-in-vesicle-aggregates-in-water-from-ph-responsive-microbial-glycolipid
#8
Niki Baccile, Patrick Le Griel, Sylvain Prévost, Bernd Everaert, Inge N A Van Bogaert, Sophie Roelants, Wim Soetaert
Vesicle-in-vesicle self-assembled containers, or vesosomes, are promising alternatives to liposomes because of their possible hierarchical encapsulation and high stability. We report herein the first example of sugar-based vesicles-in-vesicles, which we baptize glucosomes. These were prepared by using a natural microbial glycolipid (branched C22 sophorolipid) extracted from the culture medium of the yeast Pseudohyphozyma bogoriensis. Glucosomes spontaneously formed in water between pH 6 and pH 4 at room temperature, without the requirement of any additive...
August 2017: ChemistryOpen
https://www.readbyqxmd.com/read/28759578/heterologous-expression-of-the-antimyotoxic-protein-dm64-in-pichia-pastoris
#9
Saulo Martins Vieira, Surza Lucia Gonçalves da Rocha, Ana Gisele da Costa Neves-Ferreira, Rodrigo Volcan Almeida, Jonas Perales
Snakebite envenomation is a neglected condition that constitutes a public health problem in tropical and subtropical countries, including Brazil. Interestingly, some animals are resistant to snake envenomation due to the presence of inhibitory glycoproteins in their serum that target toxic venom components. DM64 is an acidic glycoprotein isolated from Didelphis aurita (opossum) serum that has been characterized as an inhibitor of the myotoxicity induced by bothropic toxins bearing phospholipase A2 (PLA2) structures...
July 2017: PLoS Neglected Tropical Diseases
https://www.readbyqxmd.com/read/28735452/characterization-of-polymeric-substance-classes-in-cereal-based-beverages-using-asymmetrical-flow-field-flow-fractionation-with-a-multi-detection-system
#10
Georg Krebs, Thomas Becker, Martina Gastl
Cereal-based beverages contain a complex mixture of various polymeric macromolecules including polysaccharides, peptides, and polyphenols. The molar mass of polymers and their degradation products affect different technological and especially sensory parameters of beverages. Asymmetrical flow field-flow fractionation (AF4) coupled with multi-angle light scattering (MALS) and refractive index detection (dRI) or UV detection (UV) is a technique for structure and molar mass distribution analysis of macromolecules commonly used for pure compound solutions...
July 22, 2017: Analytical and Bioanalytical Chemistry
https://www.readbyqxmd.com/read/28735024/n-linked-glycosylation-of-the-p24-family-protein-p24%C3%AE-5-modulates-retrograde-golgi-to-er-transport-of-k-hdel-ligands-in-arabidopsis
#11
Noelia Pastor-Cantizano, María Jesús García-Murria, Cesar Bernat-Silvestre, María Jesús Marcote, Ismael Mingarro, Fernando Aniento
The K/HDEL receptor ERD2 mediates the transport of soluble endoplasmic reticulum (ER)-resident proteins containing a C-terminal K/HDEL signal from the Golgi apparatus back to the ER via COPI (COat Protein I)-coated vesicles. Sorting of ERD2 within COPI vesicles is facilitated by p24 proteins. In Arabidopsis, p24δ5 has been shown to interact directly with ERD2 via its luminal GOLD (GOLgi Dynamics) domain and with COPI proteins via its cytoplasmic C-terminal tail at the acidic pH of the Golgi apparatus. Several members of the p24 family in mammals and yeast have been shown to be glycosylated, but whether Arabidopsis p24 proteins are glycosylated and the role of the sugar moiety in p24 function remain unclear...
August 7, 2017: Molecular Plant
https://www.readbyqxmd.com/read/28728943/production-and-biophysical-characterization-of-a-mini-membrane-protein-ost4v23d-a-functionally-important-mutant-of-yeast-oligosaccharyltransferase-subunit-ost4p
#12
Bharat Chaudhary, Suman Mazumder, Smita Mohanty
N-linked glycosylation of proteins is an essential and highly conserved co- and post-translational protein modification reaction that occurs in all eukaryotes. Oligosaccharyltransferase (OST), a multi-subunit membrane-associated enzyme complex, carries out this reaction. In the central reaction, a carbohydrate group is transferred to the side chain of a consensus asparagine residue in the newly synthesized protein. Genetic defects in humans cause a series of disorders known as congenital disorders of glycosylation (CDG) that include mental retardation, developmental delay, hypoglycemia etc...
November 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28694218/constitutive-expression-of-human-gastric-lipase-in-pichia-pastoris-and-site-directed-mutagenesis-of-key-lid-stabilizing-residues
#13
Laura Sams, Sawsan Amara, Almahdi Chakroun, Sébastien Coudre, Julie Paume, Jacqueline Giallo, Frédéric Carrière
The cDNA encoding human gastric lipase (HGL) was integrated into the genome of Pichia pastoris using the pGAPZα A transfer vector. The HGL signal peptide was replaced by the yeast α-factor to achieve an efficient secretion. Active rHGL was produced by the transformed yeast but its levels and stability were dependent on the pH. The highest activity was obtained upon buffering the culture medium at pH5, a condition that allowed preserving enzyme activity over time. A large fraction (72±2%) of secreted rHGL remained however bound to the yeast cells, and was released by washing the cell pellet with an acid glycine-HCl buffer (pH2...
October 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28688204/enhancing-expression-of-the-pseudorabies-virus-glycoprotein-e-in-yeast-and-its-application-in-an-indirect-sandwich-elisa
#14
C-Y Wu, C-W Wu, C-M Liao, M-S Chien, C Huang
AIMS: The purpose of this study was to produce a recombinant pseudorabies virus (PRV) glycoprotein E (gE) protein with the correct antigenicity for use as a low-cost diagnostic antigen. METHODS AND RESULTS: The gene fragment encoding the amino-terminal immunodominant region of PRV gE (codons 31-270) (gEN31-270) was codon optimized and expressed constitutively and secreted using a Pichia pastoris expression system. Yeast-expressed gEN31-270 (ygEN31-270) was harvested from the culture supernatant, and ygEN31-270 was shown to exhibit N-linked glycosylation...
July 8, 2017: Journal of Applied Microbiology
https://www.readbyqxmd.com/read/28669180/polysaccharides-and-oligosaccharides-produced-on-malvar-wines-elaborated-with-torulaspora-delbrueckii-cli-918-and-saccharomyces-cerevisiae-cli-889-native-yeasts-from-d-o-vinos-de-madrid
#15
Margarita García, Rafael Apolinar-Valiente, Pascale Williams, Braulio Esteve-Zarzoso, Teresa Arroyo, Julia Crespo, Thierry Doco
Polysaccharides and oligosaccharides released into Malvar white wines elaborated through pure, mixed, and sequential cultures with Torulaspora delbrueckii CLI 918 and Saccharomyces cerevisiae CLI 889 native yeasts from D.O. "Vinos de Madrid" were studied. Both fractions from different white wines were separated by high-resolution size-exclusion chromatography. Glycosyl composition and wine polysaccharide linkages were determined by GC-EI-MS chromatography. Molar-mass distributions were determined by SEC-MALLS, and intrinsic viscosity was determined by differential viscometer...
August 9, 2017: Journal of Agricultural and Food Chemistry
https://www.readbyqxmd.com/read/28651914/phenylsulfonylfuroxan-no-donor-phenols-synthesis-and-multifunctional-activities-evaluation
#16
Yundong Xie, Yaping Yang, Sen Li, Yanhong Xu, Wenfang Lu, Zizhang Chen, Guangde Yang, Yiping Li, Yongxiao Cao, Xiaoli Bian
Phenylsulfonyfuroxan nitric oxide (NO)-donor phenols were designed, synthesized and evaluated. The compounds were designed through a symbiotic approach using selected phenols and phenylsulfonylfuroxan NO-donor. The antioxidant activities of the hybrid compounds T2-T6 showed to be good in vivo. Compounds T4 and T6 revealed excellent yeast α-glucosidase inhibitory activity and anti-glycosylation activity. All of the compounds exhibited strong NO releasing activity and significant anti-platelet aggregation activity...
August 15, 2017: Bioorganic & Medicinal Chemistry
https://www.readbyqxmd.com/read/28616799/dolichol-phosphate-mannose-synthase-a-glycosyltransferase-with-unity-in-molecular-diversities
#17
REVIEW
Dipak K Banerjee, Zhenbo Zhang, Krishna Baksi, Jesús E Serrano-Negrón
N-glycans provide structural and functional stability to asparagine-linked (N-linked) glycoproteins, and add flexibility. Glycan biosynthesis is elaborative, multi-compartmental and involves many glycosyltransferases. Failure to assemble N-glycans leads to phenotypic changes developing infection, cancer, congenital disorders of glycosylation (CDGs) among others. Biosynthesis of N-glycans begins at the endoplasmic reticulum (ER) with the assembly of dolichol-linked tetra-decasaccharide (Glc3Man9GlcNAc2-PP-Dol) where dolichol phosphate mannose synthase (DPMS) plays a central role...
August 2017: Glycoconjugate Journal
https://www.readbyqxmd.com/read/28608761/biomedical-applications-of-yeast-a-patent-view-part-one-yeasts-as-workhorses-for-the-production-of-therapeutics-and-vaccines
#18
REVIEW
Farzin Roohvand, Mehdi Shokri, Meghdad Abdollahpour-Alitappeh, Parastoo Ehsani
Yeasts, as Eukaryotes, offer unique features for ease of growth and genetic manipulation possibilities, making it an exceptional microbial host. Areas covered: This review provides general and patent-oriented insights into production of biopharmaceuticals by yeasts. Patents, wherever possible, were correlated to the original or review articles. The review describes applications of major GRAS (generally regarded as safe) yeasts for the production of therapeutic proteins and subunit vaccines; additionally, immunomodulatory properties of yeast cell wall components were reviewed for use of whole yeast cells as a new vaccine platform...
August 2017: Expert Opinion on Therapeutic Patents
https://www.readbyqxmd.com/read/28598353/cellular-consequences-of-diminished-protein-o-mannosyltransferase-activity-in-baker-s-yeast
#19
Ewa Zatorska, Lihi Gal, Jaro Schmitt, Daniela Bausewein, Maya Schuldiner, Sabine Strahl
O-Mannosylation is a type of protein glycosylation initiated in the endoplasmic reticulum (ER) by the protein O-mannosyltransferase (PMT) family. Despite the vital role of O-mannosylation, its molecular functions and regulation are not fully characterized. To further explore the cellular impact of protein O-mannosylation, we performed a genome-wide screen to identify Saccharomyces cerevisiae mutants with increased sensitivity towards the PMT-specific inhibitor compound R3A-5a. We identified the cell wall and the ER as the cell compartments affected most upon PMT inhibition...
June 9, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/28597972/a-novel-interacting-protein-serp1-regulates-the-n-linked-glycosylation-and-function-of-glp-1-receptor-in-the-liver
#20
Yuanyuan Xiao, Junfeng Han, Qianqian Wang, Yueqin Mao, Meilin Wei, Weiping Jia, Li Wei
Glucagon-like peptide 1 (GLP-1) exerts multiple effects on metabolism through its receptor, GLP-1R, in the liver. Activation and transduction of GLP-1R require complex interactions of largely unknown accessory proteins, and these processes are crucial to the response to endoplasmic reticulum (ER) stress. Using the membrane-based split ubiquitin yeast two-hybrid system (MYTH) and a human liver cDNA library, we obtained the human GLP-1R interactome and identified SERP1 as a potential interacting protein based on its ability to stabilize membrane proteins and facilitate N-linked glycosylation...
June 9, 2017: Journal of Cellular Biochemistry
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