scholarly journals Expression of GA Coat Protein-Derived Mosaic Virus-Like Particles in Saccharomyces cerevisiae and Packaging in vivo of mRNAs into Particles

2012 ◽  
Vol 66 (6) ◽  
pp. 234-241
Author(s):  
Arnis Strods ◽  
Dagnija Argule ◽  
Indulis Cielens ◽  
Ludmila Jackeviča ◽  
Regīna Renhofa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Coat Protein ◽  
Pichia Pastoris ◽  
Mosaic Virus ◽  
Expression Vector ◽  
Expression Systems ◽  
Coding Sequences ◽  
Yeast Pichia Pastoris ◽  
Virus Like Particles

Our previous research showed that the best yield of virus-like particles (VLPs) formed by RNA bacteriophage GA coat protein was obtained by expression in yeast Pichia pastoris, while other used expression systems in Saccharomyces cerevisiae gave much lower amounts of capsids. The main reasons to attempt further studies in Saccharomyces cerevisiae were to improve the yield of GA-based VLPs using constructs with optimised nucleotide triplets in coding sequences, and to exploit the possibilities of the two-promoter Gal1/Gal10 system of expression vector pESC-URA for production of the desired mosaic VLPs and for packaging of mRNAs into VLPs in vivo

scholarly journals Sec16 influences transitional ER sites by regulating rather than organizing COPII

2013 ◽  
Vol 24 (21) ◽  
pp. 3406-3419 ◽  
Author(s):  
Nike Bharucha ◽  
Yang Liu ◽  
Effrosyni Papanikou ◽  
Conor McMahon ◽  
Masatoshi Esaki ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Pichia Pastoris ◽  
Protein Complex ◽  
The Other ◽  
Yeast Pichia Pastoris ◽  
Functional Regions ◽  
Conserved Domain ◽  
Copii Vesicles ◽  
Negative Regulatory Role

During the budding of coat protein complex II (COPII) vesicles from transitional endoplasmic reticulum (tER) sites, Sec16 has been proposed to play two distinct roles: negatively regulating COPII turnover and organizing COPII assembly at tER sites. We tested these ideas using the yeast Pichia pastoris. Redistribution of Sec16 to the cytosol accelerates tER dynamics, supporting a negative regulatory role for Sec16. To evaluate a possible COPII organization role, we dissected the functional regions of Sec16. The central conserved domain, which had been implicated in coordinating COPII assembly, is actually dispensable for normal tER structure. An upstream conserved region (UCR) localizes Sec16 to tER sites. The UCR binds COPII components, and removal of COPII from tER sites also removes Sec16, indicating that COPII recruits Sec16 rather than the other way around. We propose that Sec16 does not in fact organize COPII. Instead, regulation of COPII turnover can account for the influence of Sec16 on tER sites.

scholarly journals Modification of a Ubiquitin-like Protein Paz2 Conducted Micropexophagy through Formation of a Novel Membrane Structure

2004 ◽  
Vol 15 (1) ◽  
pp. 58-70 ◽  
Author(s):  
Hiroyuki Mukaiyama ◽  
Misuzu Baba ◽  
Masako Osumi ◽  
Satoshi Aoyagi ◽  
Nobuo Kato ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pichia Pastoris ◽  
Membrane Structure ◽  
De Novo ◽  
Genetic Evidence ◽  
Membrane Formation ◽  
Modification System ◽  
Yeast Pichia Pastoris ◽  
Lysosomal Membranes ◽  
To Receive

Microautophagy is a versatile process in which vacuolar or lysosomal membranes directly sequester cytosolic targets for degradation. Recent genetic evidence suggested that microautophagy uses molecular machineries essential for macroautophagy, but the details of this process are still unknown. In this study, a ubiquitin-like protein Paz2 essential for micropexophagy in the yeast Pichia pastoris has been shown to receive modification through the function of Paz8 and Gsa7, yielding a modified form Paz2-I, similar to the ubiquitin-like lipidation of Aut7 that is essential for macroautophagy in Saccharomyces cerevisiae. We identified a novel membrane structure formed after the onset of micropexophagy, which we suggest is necessary for the sequestration of peroxisomes by the vacuole. Assembly of this newly formed membrane structure, which is followed by localization of Paz2 to it, was found to require a properly functioning Paz2-modification system. We herein show that Paz2 and its modification system conduct micropexophagy through formation of the membrane structure, which explains the convergence between micropexophagy and macroautophagy with regard to de novo membrane formation.

Assembly of bacteriophage Qβ virus-like particles in yeast Saccharomyces cerevisiae and Pichia pastoris

2006 ◽  
Vol 123 (3) ◽  
pp. 297-303 ◽  
Author(s):  
Janis Freivalds ◽  
Andris Dislers ◽  
Velta Ose ◽  
Dace Skrastina ◽  
Indulis Cielens ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pichia Pastoris ◽  
Yeast Saccharomyces Cerevisiae ◽  
Virus Like Particles

Characterization of the HCV Core Virus-like Particles Produced in the Methylotrophic Yeast Pichia pastoris

2001 ◽  
Vol 287 (1) ◽  
pp. 122-125 ◽  
Author(s):  
Nelson Acosta-Rivero ◽  
Julio C. Aguilar ◽  
Alexis Musacchio ◽  
Viviana Falcón ◽  
Ariel Viña ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Methylotrophic Yeast ◽  
Yeast Pichia Pastoris ◽  
Virus Like Particles ◽  
Methylotrophic Yeast Pichia Pastoris

Mechanism of synthesis of turnip yellow mosaic virus coat protein subgenomic RNA in vivo

Virology ◽  
1989 ◽  
Vol 171 (2) ◽  
pp. 386-393 ◽  
Author(s):  
Radhia Gargouri ◽  
Rajiv L. Joshi ◽  
John F. Bol ◽  
Suzanne Astier-Manifacier ◽  
Anne-Lise Haenni
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Yellow Mosaic Virus ◽  
Turnip Yellow Mosaic Virus ◽  
Subgenomic Rna ◽  
Virus Coat Protein ◽  
Virus Coat

scholarly journals In Vivo Packaging of mRNA in Yeast-Produced Bacteriophage GA Derived Virus-Like Particles

2017 ◽  
Vol 71 (4) ◽  
pp. 266-274
Author(s):  
Dagnija Ārgule ◽  
Indulis Cielēns ◽  
Regīna Renhofa ◽  
Arnis Strods
Keyword(s):  
Coat Protein ◽  
Self Assembly ◽  
Rna Binding ◽  
Single Point ◽  
Point Mutations ◽  
Coat Protein Sequence ◽  
Yeast Cells ◽  
Virus Like Particles ◽  
Binding Characteristics

Abstract Bacteriophage GA coat protein formed self-assembly competent virus-like particles (VLPs) have been expressed previously in bacterial and yeast cells. On the basis of our previous experiments on the yeast vector pESC-URA / S. cerevisiae system containing two oppositely oriented promoters, new constructions were created with point-mutations in coat protein to mimic phage MS2-like RNA binding characteristics. Simultaneously, the MS2 operator sequence was added to mRNA desired for packaging. After the introduction of single-point mutations (S87N, K55N, R43K) and double-point mutations (S87N + K55N and S87N + R43K), the coat protein’s ability to form VLPs was retained, but yield from cells was decreased. Exchange of the 87th Ser to Asn in coat protein sequence in combination with bacteriophage MS2 translational operator provided specific packaging of the gene of interest (GFP). Although non-specific nucleic acid sequences were packaged, the remarkable specificity for packaging of the gene of interest can be achieved using the described approach.

scholarly journals Constitutive Optimized Production of Streptokinase inSaccharomyces cerevisiaeUtilizing Glyceraldehyde 3-Phosphate Dehydrogenase Promoter ofPichia pastoris

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Ravi N. Vellanki ◽  
Ravichandra Potumarthi ◽  
Kiran K. Doddapaneni ◽  
Naveen Anubrolu ◽  
Lakshmi N. Mangamoori
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pichia Pastoris ◽  
Expression Vector ◽  
Recombinant Expression ◽  
Expression System ◽  
Nitrogen Sources ◽  
Clot Lysis ◽  
Process Conditions ◽  
Scale Production ◽  
Activation Assay

A novel expression vector constructed from genes ofPichia pastoriswas applied for heterologous gene expression inSaccharomyces cerevisiae. Recombinant streptokinase (SK) was synthesized by cloning the region encoding mature SK under the control of glyceraldehyde 3-phosphate dehydrogenase (GAP) promoter ofPichia pastorisinSaccharomyces cerevisiae. SK was intracellularly expressed constitutively, as evidenced by lyticase-nitroanilide and caseinolytic assays. The functional activity was confirmed by plasminogen activation assay andin vitroclot lysis assay. Stability and absence of toxicity to the host with the recombinant expression vector as evidenced by southern analysis and growth profile indicate the application of this expression system for large-scale production of SK. Two-stage statistical approach, Plackett-Burman (PB) design and response surface methodology (RSM) was used for SK production medium optimization. In the first stage, carbon and organic nitrogen sources were qualitatively screened by PB design and in the second stage there was quantitative optimization of four process variables, yeast extract, dextrose, pH, and temperature, by RSM. PB design resulted in dextrose and peptone as best carbon and nitrogen sources for SK production. RSM method, proved as an efficient technique for optimizing process conditions which resulted in 110% increase in SK production, 2352 IU/mL, than for unoptimized conditions.

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