scholarly journals Scaffolded Antigens in Yeast Cell Particle Vaccines Provide Protection against Systemic Polyoma Virus Infection

2016 ◽  
Vol 2016 ◽  
pp. 1-15 ◽  
Author(s):  
Donald J. Tipper ◽  
Eva Szomolanyi-Tsuda
Keyword(s):  
Yeast Cell ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Subcutaneous Administration ◽  
Polyoma Virus ◽  
Apolipoprotein A1 ◽  
Yeast Cells ◽  
Scale Production ◽  
Protein Antigens ◽  
Virus Challenge

Background. U65, a self-aggregating peptide scaffold, traps fused protein antigens in yeast cells. Conversion to Yeast Cell Particle (YCP) vaccines by partial removal of surface mannoproteins exposesβ-glucan, mediating efficient uptake by antigen-presenting cells (APCs). YCP vaccines are inexpensive, capable of rapid large-scale production and have potential for both parenteral and oral use.Results. YCP processing by alkaline hydrolysis exposes up to 20% of the glucan but converts scaffolded antigen and internal yeast proteins into a common aggregate, preventing selective yeast protein removal. For U65-green fluorescent protein (GFP) or U65-Apolipoprotein A1 (ApoA1) subcutaneous vaccines, maximal IgG responses in mice required 10% glucan exposure. IgG responses to yeast proteins were 5-fold lower. Proteolytic mannoprotein removal produced YCPs with only 6% glucan exposure, insufficiently porous for selective removal of even native yeast proteins. Vaccine efficacy was reduced 10-fold. Current YCP formulations, therefore, are not suitable for human use but have considerable potential for use in feed animal vaccines. Significantly, a YCP vaccine expressing a GFP fusion to VP1, the murine polyoma virus major capsid protein, after either oral or subcutaneous administration, protected mice against an intraperitoneal polyoma virus challenge, reducing viral DNA levels in spleen and liver by >98%.

scholarly journals Yeast Actin-Related Protein ARP6 Negatively RegulatesAgrobacterium-Mediated Transformation of Yeast Cell

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Yumei Luo ◽  
Zikai Chen ◽  
Detu Zhu ◽  
Haitao Tu ◽  
Shen Quan Pan
Keyword(s):  
Genetic Engineering ◽  
Large Scale ◽  
Transformation Efficiency ◽  
Genetic Diseases ◽  
Negative Regulator ◽  
Yeast Cells ◽  
Scale Production ◽  
Related Protein ◽  
Cellular Mechanisms ◽  
Eukaryotic Organisms

The yeasts, includingSaccharomyces cerevisiaeandPichia pastoris, are single-cell eukaryotic organisms that can serve as models for human genetic diseases and hosts for large scale production of recombinant proteins in current biopharmaceutical industry. Thus, efficient genetic engineering tools for yeasts are of great research and economic values.Agrobacterium tumefaciens-mediated transformation (AMT) can transfer T-DNA into yeast cells as a method for genetic engineering. However, how the T-DNA is transferred into the yeast cells is not well established yet. Here our genetic screening of yeast knockout mutants identified a yeast actin-related proteinARP6as a negative regulator of AMT.ARP6is a critical member of the SWR1 chromatin remodeling complex (SWR-C); knocking out some other components of the complex also increased the transformation efficiency, suggesting thatARP6might regulate AMT via SWR-C. Moreover, knockout ofARP6led to disruption of microtubule integrity, higher uptake and degradation of virulence proteins, and increased DNA stability inside the cells, all of which resulted in enhanced transformation efficiency. Our findings have identified molecular and cellular mechanisms regulating AMT and a potential target for enhancing the transformation efficiency in yeast cells.

scholarly journals Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection

Blood ◽  
2009 ◽  
Vol 113 (21) ◽  
pp. 5104-5110 ◽  
Author(s):  
Robert E. Throm ◽  
Annastasia A. Ouma ◽  
Sheng Zhou ◽  
Anantharaman Chandrasekaran ◽  
Timothy Lockey ◽  
...  
Keyword(s):  
Cell Lines ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Lentiviral Vector ◽  
Cell Clone ◽  
Severe Combined Immunodeficiency ◽  
Interleukin 2 ◽  
Retroviral Vectors ◽  
Scale Production ◽  
Producer Cell

AbstractRetroviral vectors containing internal promoters, chromatin insulators, and self-inactivating (SIN) long terminal repeats (LTRs) may have significantly reduced genotoxicity relative to the conventional retroviral vectors used in recent, otherwise successful clinical trials. Large-scale production of such vectors is problematic, however, as the introduction of SIN vectors into packaging cells cannot be accomplished with the traditional method of viral transduction. We have derived a set of packaging cell lines for HIV-based lentiviral vectors and developed a novel concatemeric array transfection technique for the introduction of SIN vector genomes devoid of enhancer and promoter sequences in the LTR. We used this method to derive a producer cell clone for a SIN lentiviral vector expressing green fluorescent protein, which when grown in a bioreactor generated more than 20 L of supernatant with titers above 107 transducing units (TU) per milliliter. Further refinement of our technique enabled the rapid generation of whole populations of stably transformed cells that produced similar titers. Finally, we describe the construction of an insulated, SIN lentiviral vector encoding the human interleukin 2 receptor common γ chain (IL2RG) gene and the efficient derivation of cloned producer cells that generate supernatants with titers greater than 5 × 107 TU/mL and that are suitable for use in a clinical trial for X-linked severe combined immunodeficiency (SCID-X1).

scholarly journals Development of plant regeneration and Agrobacterium tumefaciens-mediated transformation methodology for Physalis pruinosa

2018 ◽  
Author(s):  
Kerry Swartwood ◽  
Joyce Van Eck
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plant Regeneration ◽  
Genetic Engineering ◽  
Large Scale ◽  
Gene Editing ◽  
Fluorescent Protein ◽  
Pcr Analysis ◽  
Marker Genes ◽  
Scale Production ◽  
Hypocotyl Explants

AbstractPhysalis pruinosa, also known as groundcherry, produces a small, yellow, highly nutritious edible fruit that is enveloped by a papery husk. In order for the potential of large-scale production of P. pruinosa fruit to be realized, undesirable characteristics, such as an unmanageable, sprawling growth habit and extensive fruit drop, need to be improved by exploiting approaches available through plant breeding, genetic engineering, and gene editing. In this study, we established plant regeneration and Agrobacterium tumefaciens-mediated methods to allow application of genetic engineering and gene editing of P. pruinosa. Cotyledon and hypocotyl explants from 7 – 8-day-old in vitro-grown seedlings were assessed for plant regeneration. Explants were cultured for 2 weeks on a Murashige and Skoog salts-based medium that contained 2 mg/L zeatin followed by transfer to medium containing 1 mg/L zeatin. Only hypocotyl explants regenerated shoots. Hypocotyl explants were infected with Agrobacterium tumefaciens strain AGL1 containing the pJL33 binary vector that has the green fluorescent protein (GFP) reporter and neomycin phosphotransferase II (nptII) selectable marker genes. After cocultivation, explants were cultured on selective plant regeneration medium that contained 50, 100, 200, 250, and 300 mg/L kanamycin to determine the most effective level for efficient recovery of transgenic lines. Based on rooting of regenerated shoots on selective medium, GFP visualization, and PCR analysis for the presence of the nptII gene, medium containing 200 mg/L kanamycin resulted in the highest transformation efficiency at 24%. This study sets the foundation for future genetic engineering and gene editing approaches for improvement of P. pruinosa.

scholarly journals A Novel Method for High-Level Production of TEV Protease by Superfolder GFP Tag

2009 ◽  
Vol 2009 ◽  
pp. 1-8 ◽  
Author(s):  
Xudong Wu ◽  
Di Wu ◽  
Zhisheng Lu ◽  
Wentao Chen ◽  
Xiaojian Hu ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Scale Production ◽  
Sequence Specificity ◽  
Tev Protease ◽  
Large Scale Production ◽  
Novel Method

Because of its stringent sequence specificity, tobacco etch virus (TEV) protease is widely used to remove fusion tags from recombinant proteins. Due to the poor solubility of TEV protease, many strategies have been employed to increase the expression level of this enzyme. In our work, we introduced a novel method to produce TEV protease by using visible superfolder green fluorescent protein (sfGFP) as the fusion tag. The soluble production and catalytic activity of six variants ofsfGFP-TEV was examined, and then the best variant was selected for large-scale production. After purified by Ni-NTA affinity chromatography and Q anion exchange chromatography, the best variant ofsfGFP-TEV fusion protease was obtained with purity of over 98% and yield of over 320 mg per liter culture. ThesfGFP-TEV had a similar catalytic activity to that of the original TEV protease. Our research showed a novel method of large-scale production of visible and functional TEV protease for structural genomics research and other applications.

AGRICULTURAL PRODUCTION OF CA DONG PEOPLE IN RESETTLEMENT AREA OF TRANH RIVER HYDROPOWER NUMBER 2: CURRENT SITUATION AND IMPACT FACTORS

2020 ◽  
Vol 9 (2) ◽  
Author(s):  
Bùi Thị Bích Lan
Keyword(s):  
Social Relations ◽  
Agricultural Production ◽  
Large Scale ◽  
Transition Process ◽  
Impact Factors ◽  
Small Scale ◽  
Scale Production ◽  
Technical Application ◽  
Production Models ◽  
Role Of The State

In Vietnam, the construction of hydropower projects has contributed significantly in the cause of industrialization and modernization of the country. The place where hydropower projects are built is mostly inhabited by ethnic minorities - communities that rely primarily on land, a very important source of livelihood security. In the context of the lack of common productive land in resettlement areas, the orientation for agricultural production is to promote indigenous knowledge combined with increasing scientific and technical application; shifting from small-scale production practices to large-scale commodity production. However, the research results of this article show that many obstacles in the transition process are being posed such as limitations on natural resources, traditional production thinking or the suitability and effectiveness of scientific - technical application models. When agricultural production does not ensure food security, a number of implications for people’s lives are increasingly evident, such as poverty, preserving cultural identity, social relations and resource protection. Since then, it has set the role of the State in researching and building appropriate agricultural production models to exploit local strengths and ensure sustainability.

I. S. A. Baud. Forms of Production and Women's Labour: Gender Aspects of Industrialisation in India and Mexico. New Delhi: Sage Publications. 1992. 321 pp.Hardbound. Price: Indian Rs 295.00.

1993 ◽  
Vol 32 (1) ◽  
pp. 129-131
Author(s):  
Naureen Talha
Keyword(s):  
Large Scale ◽  
Indian Society ◽  
New Delhi ◽  
Scale Production ◽  
Female Labour ◽  
Self Employment ◽  
Commodity Production ◽  
Large Scale Production ◽  
Mexican Society ◽  
Small Production

The literature on female labour in Third World countries has become quite extensive. India, being comparatively more advanced industrially, and in view of its size and population, presents a pictures of multiplicity of problems which face the female labour market. However, the author has also included Mexico in this analytical study. It is interesting to see the characteristics of developing industrialisation in two different societies: the Indian society, which is conservative, and the Mexican society, which is progressive. In the first chapter of the book, the author explains that he is not concerned with the process of industrialisation and female labour employed at different levels of work, but that he is interested in forms of production and women's employment in large-scale production, petty commodity production, marginal small production, and self-employment in the informal sector. It is only by analysis of these forms that the picture of females having a lower status is understood in its social and political setting.

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