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yeast glycosylation

Nooshin Mohandesi, Seyed Omid Ranaei Siadat, Kamahldin Haghbeen, Ardeshir Hesampour
Invertase (EC. catalyzes the hydrolysis of sucrose to an equimolar mixture of D-glucose and D-fructose which is of interest for various industrial applications. In this research, Saccharomyces cerevisiae invertase gene (SUC2) was optimized based on Pichia pastoris codon preference. The synthetic gene was introduced into the methylotrophic yeast Pichia pastoris under the control of the inducible AOX1 promoter. High level of the extracellular recombinant invertase (R-inv) production was achieved via methanol induction for 4 days and purified by His-Tag affinity chromatography which appeared to be a mixture of glycosylated proteins with various sizes of 85-95 kDa on SDS-PAGE...
December 2016: 3 Biotech
Sathya Prabhu, Shanmugam Vinodhini, Chakravarthy Elenchezhiyan, Devi Rajeswari
BACKGROUND: Medicinal plants and green synthesis of silver nanoparticles (AgNPs) have proven to be potent source effective agents in the treatment of diabetes mellitus. The present study is focused on the green synthesis of AgNPs from aqueous leaf extract of Pouteria sapota (P. sapota) in order to evaluate the in-vitro and in-vivo anti-diabetic property of aqueous extracts and their synthesized AgNPs. METHODS: AgNPs were biologically synthesized under ambient conditions from an aqueous leaf extract of P...
March 21, 2017: Journal of Diabetes
Dagmar Schlenzig, Stephan Schilling
Meprins are zinc-dependent proteases of the metzincin superfamily of metalloproteases. The enzymes are extracellular multi-domain proteins which are stabilized by disulfide bridges, dimerization, and glycosylation. Due to their complex structure, recombinant expression was first established in mammalian and insect cells. However, these methods have several disadvantages such as high costs and the low yields. For this reason, yeast is often considered a preferable expression system. Here, we describe the manipulation and secretory expression of human meprin β in the methylotrophic yeast P...
2017: Methods in Molecular Biology
Yasuo Mitani, Yoshimi Oshima, Nobutaka Mitsuda, Azusa Tomioka, Masako Sukegawa, Mika Fujita, Hiroyuki Kaji, Yoshihiro Ohmiya
Cypridina noctiluca luciferase has been utilized for biochemical and molecular biological applications, including bioluminescent enzyme immunoassays, far-red luminescence imaging, and high-throughput reporter assays. Some of these applications require a large amount of purified luciferase. However, conventional protein expression systems are not capable of producing sufficient quantities of protein with a high quality and purity without laborious and costly purification processes. To improve the productivity and expand the breadth of possibilities for Cypridina luciferase applications, we employed a variety of secretion expression systems, including yeast, mammalian cells, and silk worms...
March 11, 2017: Protein Expression and Purification
Timo Johannssen, Bernd Lepenies
Glycosylation is an integral post-translational modification present in more than half of all eukaryotic proteins. It affects key protein functions, including folding, stability, and immunogenicity. Glycoengineering approaches, such as the use of bacterial N-glycosylation systems, or expression systems, including yeasts, insect cells, and mammalian cells, have enabled access to defined and homogenous glycoproteins. Given that glycan structures on proteins can be recognized by host lectin receptors, they may facilitate cell-specific targeting and immune modulation...
October 27, 2016: Trends in Biotechnology
Xue-Qing Yang, Wei Wang, Xiao-Ling Tan, Xiao-Qi Wang, Hui Dong
On the basis of prior work, cytochrome P450 CYP9A61 was found to be enriched in fat bodies and during feeding stages, and transcription was induced by λ-cyhalothrin in Cydia pomonella. In this study, recombinant CYP9A61 was expressed in Escherichia coli and Pichia pastoris, and its biochemical properties were investigated. Substrate saturation curves and biochemical properties revealed that, in the presence of glycosylation, the yeast-secreted CYP9A61 exhibited a higher affinity for the substrate p-nitroanisole and was found to be more stable at certain pHs and temperatures than bacterially produced CYP9A61...
March 14, 2017: Journal of Agricultural and Food Chemistry
Xiao-Mei Huang, Qing-Qing Li, Xiu-Ling Chen, Jin-Xia Fan, Xiu-Hong Xu, Xu-Dong Sun, Dong-Yu Li, Hong-Xiao Zhao
Endoglucanase secreted by the fungus Trichoderma atroviride is a kind of cellulase. An endoglucanase gene egII was cloned from T. atroviride AS3.3013 and expressed in Saccharomyces cerevisiae INVScI. The open reading frame of the egII gene was composed of 1257 bp, encoding 418 amino acids with a molecular weight of 44.23 kDa plus a signal peptide of 21 amino acids. Based on sequence similarity, TaEGII belonged to the glycosyl hydrolase family 5. Expression of the egII gene in T. atroviride AS3.3013 can be induced by microcrystalline cellulose (MCC), bran, carboxymethyl cellulose (CMC), rice straw, and corn stalk but is inhibited by glucose...
February 2, 2017: Applied Biochemistry and Biotechnology
Andrew L Garfoot, Kacey L Dearing, Andrew D VanSchoiack, Vicki H Wysocki, Chad A Rappleye
Fungal cell walls contain β-glucan polysaccharides which stimulate immune responses when recognized by host immune cells. The fungal pathogen Histoplasma capsulatum minimizes detection of β-glucan by host cells through at least two mechanisms: concealment of β-glucans beneath α-glucans and enzymatic removal of any exposed β-glucan polysaccharides by the secreted glucanase Eng1. Histoplasma yeasts also secrete the putative glucanase Exg8, which may serve a similar role as Eng1 in removing exposed β-glucans from the yeast cell surface...
February 2, 2017: Journal of Biological Chemistry
Anne-Sophie Colinet, Louise Thines, Antoine Deschamps, Gaëlle Flémal, Didier Demaegd, Pierre Morsomme
The UPF0016 family is a recently identified group of poorly characterized membrane proteins whose function is conserved through evolution and that are defined by the presence of one or two copies of the E-ϕ-G-D-[KR]-[TS] consensus motif in their transmembrane domain. We previously showed that two members of this family, the human TMEM165 and the budding yeast Gdt1p, are functionally related and are likely to form a new group of Ca(2+) transporters. Mutations in TMEM165 have been demonstrated to cause a new type of rare human genetic diseases denominated as Congenital Disorders of Glycosylation...
January 23, 2017: Cellular Microbiology
Germán G Kaiser, Nicolás C Mucci, Vega González, Lourdes Sánchez, José A Parrón, María D Pérez, Miguel Calvo, Juan F Aller, Federico A Hozbor, Adrián A Mutto
Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection...
March 2017: Journal of Dairy Science
Ken Ishikawa, Scott H Medina, Joel P Schneider, Amar J S Klar
Although resistance toward small-molecule chemotherapeutics has been well studied, the potential of tumor cells to avoid destruction by membrane-lytic compounds remains unexplored. Anticancer peptides (ACPs) are a class of such agents that disrupt tumor cell membranes through rapid and non-stereospecific mechanisms, encouraging the perception that cellular resistance toward ACPs is unlikely to occur. We demonstrate that eukaryotic cells can, indeed, develop resistance to the model oncolytic peptide SVS-1, which preferentially disrupts the membranes of cancer cells...
February 16, 2017: Cell Chemical Biology
Gabriela Llauger, Luis Alejandro de Haro, Victoria Alfonso, Mariana Del Vas
Mal de Río Cuarto virus (MRCV) is a member of the Fijivirus genus, within the Reoviridae family, that replicates and assembles in cytoplasmic inclusion bodies called viroplasms. In this study, we investigated interactions between ten MRCV proteins by yeast two-hybrid (Y2H) assays and identified interactions of non-structural proteins P6/P6, P9-2/P9-2 and P6/P9-1. P9-1 and P6 are the major and minor components of the viroplasms respectively, whereas P9-2 is an N-glycosylated membrane protein of unknown function...
January 10, 2017: Virus Research
Christoph Kreer, Janina M Kuepper, Matthias Zehner, Thomas Quast, Waldemar Kolanus, Beatrix Schumak, Sven Burgdorf
N-glycosylation is generally accepted to enhance the immunogenicity of antigens because of two main reasons. First, the attachment of glycans enables recognition by endocytic receptors like the mannose receptor (MR) and hence increased uptake by dendritic cells (DCs). Second, foreign glycans are postulated to be immunostimulatory and their recognition could induce DC activation. However, a direct comparison between the immunogenicity of N-glycosylated vs. de-glycosylated proteins in vivo and a direct effect of N-glycosylated antigens on the intrinsic capacity of DCs to activate T cells have not been assessed so far...
January 24, 2017: Oncotarget
Genny Degani, Alessandra A Altomare, Mara Colzani, Caterina Martino, Angelica Mazzolari, Guenter Fritz, Giulio Vistoli, Laura Popolo, Giancarlo Aldini
The Advanced Glycation and Lipoxidation End products (AGEs and ALEs) are a heterogeneous class of compounds derived from the non-enzymatic glycation or protein adduction by lipoxidation break-down products. The receptor for AGEs (RAGE) is involved in the progression of chronic diseases based on persistent inflammatory state and oxidative stress. RAGE is a pattern recognition receptor (PRR) and the inhibition of the interaction with its ligands or of the ligand accumulation have a potential therapeutic effect...
April 2017: Redox Biology
Weixiong Hu, Xiaoyun Liu, Yufeng Li, Daling Liu, Zhihe Kuang, Chuiwen Qian, Dongsheng Yao
β-Mannanase has been widely used in industries such as food and feed processing and thus has been a target enzyme for biotechnological development. In this study, we sought to improve the stability and protease resistance of a recombinant β-mannanase, MAN47 from Armillariella tabescens, through rationally designed N-glycosylation. Based on homology modeling, molecular docking, secondary structure analysis and glycosylation feasibility analysis, an enhanced aromatic sequon sequence was introduced into specific MAN47 loop regions to facilitate N-glycosylation...
February 2017: Enzyme and Microbial Technology
Bipasha Bhattacharjee, Neeta Pathaw, Nikhil K Chrungoo, Atanu Bhattacharjee
Chitinases are varied sized proteins which have the ability to degrade chitin and are present in a huge range of organisms like fungi, yeasts, arthropods, humans etc. and have been getting increased attention due to their biocontrol properties. In silico analysis sheds light on the extensive properties of this plant protein. In this paper, a particular antifungal protein Chitinase sourced from Sechium edule from East Khasi Hills, Meghalaya was characterized using an array of bioinformatics tools. The modelled protein showed conserved domains characteristic to glycosyl hydrolase, family 18 superfamily...
March 30, 2017: Gene
Jacob A Englaender, Yuanyuan Zhu, Abhijit N Shirke, Lei Lin, Xinyue Liu, Fuming Zhang, Richard A Gross, Mattheos A G Koffas, Robert J Linhardt
Heparin, an anticoagulant drug, is biosynthesized in selected animal cells. The heparin biosynthetic enzymes mainly consist of sulfotransferases and all are integral transmembrane glycoproteins. These enzymes are generally produced in engineered Escherichia coli as without their transmembrane domains as non-glycosylated fusion proteins. In this study, we used the yeast, Komagataella pastoris, to prepare four sulfotransferases involved in heparin biosynthesis as glycoproteins. While the yields of these yeast-expressed enzymes were considerably lower than E...
December 14, 2016: Applied Microbiology and Biotechnology
Johanna M Smeekens, Haopeng Xiao, Ronghu Wu
Protein secretion is essential for numerous cellular activities, and secreted proteins in bodily fluids are a promising and noninvasive source of biomarkers for disease detection. Systematic analysis of secreted proteins and glycoproteins will provide insight into protein function and cellular activities. Yeast (Saccharomyces cerevisiae) is an excellent model system for eukaryotic cells, but global analysis of secreted proteins and glycoproteins in yeast is challenging due to the low abundances of secreted proteins and contamination from high-abundance intracellular proteins...
December 20, 2016: Journal of Proteome Research
Petra Gottier, Amaia Gonzalez-Salgado, Anant K Menon, Yuk-Chien Liu, Alvaro Acosta-Serrano, Peter Bütikofer
The membrane protein RFT1 is essential for normal protein N-glycosylation, but its precise function is not known. RFT1 was originally proposed to translocate the glycolipid Man5GlcNAc2-PP-dolichol (needed to synthesize N-glycan precursors) across the endoplasmic reticulum membrane, but subsequent studies showed that it does not play a direct role in transport. In contrast to the situation in yeast, RFT1 is not essential for growth of the parasitic protozoan Trypanosoma brucei, enabling the study of its function in a null background...
January 20, 2017: Journal of Biological Chemistry
Jeffrey M Hollomon, Nora Grahl, Sven D Willger, Katja Koeppen, Deborah A Hogan
Candida albicans behaviors are affected by pH, an important environmental variable. Filamentous growth is a pH-responsive behavior, where alkaline conditions favor hyphal growth and acid conditions favor growth as yeast. We employed filamentous growth as a tool to study the impact of pH on the hyphal growth regulator Cyr1, and we report that downregulation of cyclic AMP (cAMP) signaling by acidic pH contributes to the inhibition of hyphal growth in minimal medium with GlcNAc. Ras1 and Cyr1 are generally required for efficient hyphal growth, and the effects of low pH on Ras1 proteolysis and GTP binding are consistent with diminished cAMP output...
November 2016: MSphere
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