scholarly journals A method for the generation of pseudotyped virus particles bearing SARS coronavirus spike protein in high yields

2021 ◽  
Author(s):  
Yoichiro Fujioka ◽  
Sayaka Kashiwagi ◽  
Aiko Yoshida ◽  
Aya O. Satoh ◽  
Mari Fujioka ◽  
...  
Keyword(s):  
Global Economy ◽  
Expression System ◽  
Antiviral Agents ◽  
Production Method ◽  
Spike Protein ◽  
High Yield ◽  
Pseudotyped Virus ◽  
Efficient Production ◽  
Live Virus ◽  
High Yields

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has threatened human health and the global economy. Development of additional vaccines and therapeutics is urgently required, but such development with live virus must be conducted with biosafety level 3 confinement. Pseudotyped viruses have been widely adopted for studies of virus entry and pharmaceutical development to overcome this restriction. Here we describe a modified protocol to generate vesicular stomatitis virus (VSV) pseudotyped with SARS-CoV or SARS-CoV-2 Spike protein in high yield. We found that pseudovirions produced with the conventional transient expression system lacked coronavirus Spike protein at their surface as a result of inhibition of parental VSV infection by overexpression of this protein. Establishment of stable cell lines with an optimal expression level of coronavirus Spike protein allowed the efficient production of progeny pseudoviruses decorated with Spike protein. This improved VSV pseudovirus production method should facilitate studies of coronavirus entry and development of antiviral agents.

scholarly journals High-yield Production of Amyloid-β Peptide Enabled by a Customized Spider Silk Domain

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Axel Abelein ◽  
Gefei Chen ◽  
Kristīne Kitoka ◽  
Rihards Aleksis ◽  
Filips Oleskovs ◽  
...  
Keyword(s):  
Spider Silk ◽  
Amyloid Β ◽  
Silk Gland ◽  
High Yield ◽  
Efficient Production ◽  
Amyloid Β Peptide ◽  
Nephila Clavipes ◽  
Recombinant Production ◽  
Yield Production ◽  
High Yields

AbstractDuring storage in the silk gland, the N-terminal domain (NT) of spider silk proteins (spidroins) keeps the aggregation-prone repetitive region in solution at extreme concentrations. We observe that NTs from different spidroins have co-evolved with their respective repeat region, and now use an NT that is distantly related to previously used NTs, for efficient recombinant production of the amyloid-β peptide (Aβ) implicated in Alzheimer’s disease. A designed variant of NT from Nephila clavipes flagelliform spidroin, which in nature allows production and storage of β-hairpin repeat segments, gives exceptionally high yields of different human Aβ variants as a solubility tag. This tool enables efficient production of target peptides also in minimal medium and gives up to 10 times more isotope-labeled monomeric Aβ peptides per liter bacterial culture than previously reported.

scholarly journals Synthetic Biology for Engineering Acetyl Coenzyme A Metabolism in Yeast

mBio ◽  
2014 ◽  
Vol 5 (6) ◽  
Author(s):  
Jens Nielsen
Keyword(s):  
Coenzyme A ◽  
Pyruvate Dehydrogenase Complex ◽  
High Yield ◽  
Cell Factory ◽  
Efficient Production ◽  
Acetyl Coa ◽  
Acetyl Coenzyme A ◽  
Content Type ◽  
Acetyl Coenzyme ◽  
High Yields

ABSTRACT The yeast Saccharomyces cerevisiae is a widely used cell factory for the production of fuels, chemicals, and pharmaceuticals. The use of this cell factory for cost-efficient production of novel fuels and chemicals requires high yields and low by-product production. Many industrially interesting chemicals are biosynthesized from acetyl coenzyme A (acetyl-CoA), which serves as a central precursor metabolite in yeast. To ensure high yields in production of these chemicals, it is necessary to engineer the central carbon metabolism so that ethanol production is minimized (or eliminated) and acetyl-CoA can be formed from glucose in high yield. Here the perspective of generating yeast platform strains that have such properties is discussed in the context of a major breakthrough with expression of a functional pyruvate dehydrogenase complex in the cytosol.

scholarly journals High-yield production of functionally active human serum transferrin using a baculovirus expression system, and its structural characterization

1996 ◽  
Vol 319 (1) ◽  
pp. 191-195 ◽  
Author(s):  
Stuart A. ALI ◽  
Heidi C JOAO ◽  
Robert CSONGA ◽  
Franz HAMMERSCHMID ◽  
Alexander STEINKASSERER
Keyword(s):  
Human Serum ◽  
Expression System ◽  
Baculovirus Expression System ◽  
High Yield ◽  
Efficient Production ◽  
Serum Transferrin ◽  
Human Serum Transferrin ◽  
Mammalian Expression ◽  
Baculovirus Expression ◽  
Wide Range

Recently, there has been much interest in expressing recombinant human serum transferrin (HST) and mutants thereof for structural and functional studies. We have developed a baculovirus expression system for the rapid and efficient production of large quantities of HST (> 20 mg/l). Like native HST, the recombinant protein can bind two ferric ions in the presence of bicarbonate, and is actively taken up by receptor-mediated endocytosis. Secondary structure calculations from CD measurements indicate a content of 42% α-helix and 28% β-sheet. This is the first reported use of a non-mammalian expression system to produce functional HST, and will provide a practical tool to allow expression of a wide range of HST variants for mutagenesis studies.

scholarly journals Binding of SARS-CoV-2 spike protein to ACE2 is disabled by thiol-based drugs; evidence from in vitro SARS-CoV-2 infection studies

2020 ◽  
Author(s):  
Kritika Khanna ◽  
Wilfred Raymond ◽  
Annabelle R. Charbit ◽  
Jing Jin ◽  
Irina Gitlin ◽  
...  
Keyword(s):  
Host Cells ◽  
Surface Glycoprotein ◽  
Spike Protein ◽  
Pseudotyped Virus ◽  
Cell Infection ◽  
Live Virus ◽  
Angiotensin Converting Enzyme 2 ◽  
Novel Treatments ◽  
Binding Interface

AbstractCoronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the SARS-CoV-2 spike protein is an envelope glycoprotein that binds angiotensin converting enzyme 2 as an entry receptor. The capacity of enveloped viruses to infect host cells depends on a precise thiol/disulfide balance in their surface glycoprotein complexes. To determine if cystines in the SARS-CoV-2 spike protein maintain a native binding interface that can be disrupted by drugs that cleave cystines, we tested if thiol-based drugs have efficacy in receptor binding and cell infection assays. We found that thiol-based drugs, cysteamine and WR-1065 (the active metabolite of amifostine) in particular, decrease binding of SARS-CoV-2 spike protein to its receptor, decrease the entry efficiency of SARS-CoV-2 spike pseudotyped virus, and inhibit SARS-CoV-2 live virus infection. Our findings uncover a vulnerability of SARS-CoV-2 to thiol-based drugs and provide rationale to test thiol-based drugs, especially cysteamine and amifostine, as novel treatments for COVID-19.One Sentence SummaryThiol-based drugs decrease binding of SARS-CoV-2 spike protein to its receptor and inhibit SARS-CoV-2 cell entry.

Evaluation of germ plasm for resistant to Soybean mosaic virus (SMV)

2020 ◽  
Vol 21 (1) ◽  
pp. 6-9
Author(s):  
Wuye Ria Andayanie
Keyword(s):  
Abiotic Stress ◽  
Environmental Factors ◽  
Mosaic Virus ◽  
Polyclonal Antibody ◽  
Symptom Severity ◽  
Germ Plasm ◽  
Polyclonal Antibodies ◽  
Soybean Mosaic Virus ◽  
High Yield ◽  
High Yields

Soybean superior varieties with high yields and are resistant to abiotic stress have been largely released, although some varieties grown in the field are not resistant to SMV. In addition, the opportunity to obtain lines of hope as prospective varieties with high yield and resistance to SMV is very small. The method for evaluating soybean germplasm is based on serological observations of 98 accessions of leaf samples from SMV inoculation with T isolate. The evaluation results of 98 accessions based on visual observations showed 31 genotypes reacting very resistant or healthy to mild resistant category to SMV T isolate  with a percentage of symptom severity of 0 −30 %. Among 31 genotypes there are 2 genotypes (PI 200485; M8Grb 44; Mlg 3288) with the category of visually very resistant and resistant, respectively and  Mlg 3288  with the category of mild resistant.  They have a good agronomic appearance with a weight of 100 seeds (˃10 g) and react negatively with polyclonal antibodies to SMV, except Mlg 3288 reaction is not consistent, despite the weight of 100 seeds (˃ 10 g). Leaf samples from 98 accessions revealed various symptoms of SMV infection in the field. This diversity of symptoms is caused by susceptibility to accession, when infection occurs, and environmental factors. Keywords—: soybean; genotipe; Soybean mosaic virus (SMV); disease severity; polyclonal  antibody

scholarly journals UV Nanoimprint Lithography: Geometrical Impact on Filling Properties of Nanoscale Patterns

Nanomaterials ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 822
Author(s):  
Christine Thanner ◽  
Martin Eibelhuber
Keyword(s):  
Layer Thickness ◽  
Process Parameters ◽  
Nanoimprint Lithography ◽  
Failure Modes ◽  
Production Method ◽  
Efficient Production ◽  
Comprehensive Overview ◽  
Replication Method ◽  
Wide Range ◽  
Pattern Size

Ultraviolet (UV) Nanoimprint Lithography (NIL) is a replication method that is well known for its capability to address a wide range of pattern sizes and shapes. It has proven to be an efficient production method for patterning resist layers with features ranging from a few hundred micrometers and down to the nanometer range. Best results can be achieved if the fundamental behavior of the imprint resist and the pattern filling are considered by the equipment and process parameters. In particular, the material properties and pattern size and shape play a crucial role. For capillary force-driven filling behavior it is important to understand the influencing parameters and respective failure modes in order to optimize the processes for reliable full wafer manufacturing. In this work, the nanoimprint results obtained for different pattern geometries are compared with respect to pattern quality and residual layer thickness: The comprehensive overview of the relevant process parameters is helpful for setting up NIL processes for different nanostructures with minimum layer thickness.

scholarly journals Bombyx mori Pupae Efficiently Produce Recombinant AAV2/HBoV1 Vectors with a Bombyx mori Nuclear Polyhedrosis Virus Expression System

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 704
Author(s):  
Qian Yu ◽  
Pengfei Chang ◽  
Xiaoxuan Liu ◽  
Peng Lü ◽  
Qi Tang ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bombyx Mori ◽  
Nuclear Polyhedrosis Virus ◽  
Expression System ◽  
Efficient Production ◽  
Human Bocavirus ◽  
Adeno Associated Virus ◽  
Silkworm Pupae ◽  
Polyhedrosis Virus ◽  
Nuclear Polyhedrosis

Recombinant adeno-associated virus (AAV) vectors have broad application prospects in the field of gene therapy. The establishment of low-cost and large-scale manufacturing is now the general agenda for industry. The baculovirus-insect cell/larva expression system has great potential for these applications due to its scalability and predictable biosafety. To establish a more efficient production system, Bombyx mori pupae were used as a new platform and infected with recombinant Bombyx mori nuclear polyhedrosis virus (BmNPV). The production of a chimeric recombinant adeno-associated virus (rAAV) serotype 2/human bocavirus type-1 (HBoV1) vector was used to evaluate the efficiency of this new baculovirus expression vector (BEV)–insect expression system. For this purpose, we constructed two recombinant BmNPVs, which were named rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP. The yields of rAAV2/HBoV1 derived from the rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP co-infected BmN cells exceeded 2 × 104 vector genomes (VG) per cell. The rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP can express stably for at least five passages. Significantly, rAAV2/HBoV1 could be efficiently generated from BmNPV-infected silkworm larvae and pupae at average yields of 2.52 × 1012 VG/larva and 4.6 × 1012 VG/pupa, respectively. However, the vectors produced from the larvae and pupae had a high percentage of empty particles, which suggests that further optimization is required for this platform in the future. Our work shows that silkworm pupae, as an efficient bioreactor, have great potential for application in the production of gene therapy vectors.

scholarly journals Additive-Enhanced Exfoliation for High-Yield 2D Materials Production

Nanomaterials ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 601
Author(s):  
Dinh-Tuan Nguyen ◽  
Hsiang-An Ting ◽  
Yen-Hsun Su ◽  
Mario Hofmann ◽  
Ya-Ping Hsieh
Keyword(s):  
Large Scale ◽  
2D Materials ◽  
Transition Metal Dichalcogenides ◽  
Spectroscopic Characterization ◽  
High Yield ◽  
Nanometer Scale ◽  
Efficient Production ◽  
Device Performance ◽  
Metal Dichalcogenides ◽  
Scale Deposition

The success of van-der-Waals electronics, which combine large-scale-deposition capabilities with high device performance, relies on the efficient production of suitable 2D materials. Shear exfoliation of 2D materials’ flakes from bulk sources can generate 2D materials with low amounts of defects, but the production yield has been limited below industry requirements. Here, we introduce additive-assisted exfoliation (AAE) as an approach to significantly increase the efficiency of shear exfoliation and produce an exfoliation yield of 30%. By introducing micrometer-sized particles that do not exfoliate, the gap between rotor and stator was dynamically reduced to increase the achievable shear rate. This enhancement was applied to WS2 and MoS2 production, which represent two of the most promising 2D transition-metal dichalcogenides. Spectroscopic characterization and cascade centrifugation reveal a consistent and significant increase in 2D material concentrations across all thickness ranges. Thus, the produced WS2 films exhibit high thickness uniformity in the nanometer-scale and can open up new routes for 2D materials production towards future applications.

scholarly journals Performance of Strawberry Varieties Developed for Perennial Matted-Row Production in Annual Plasticulture in a Cold Climate Region

Agronomy ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1407
Author(s):  
Courtney A. Weber
Keyword(s):  
Large Scale ◽  
Fruit Size ◽  
Weather Conditions ◽  
Cold Climate ◽  
Annual Production ◽  
High Yield ◽  
Production Cycle ◽  
Late Season ◽  
Berry Weight ◽  
High Yields

Annual plasticulture production of strawberries promises superior weed control, fruit quality and yields. However, strawberry varieties adapted for perennial, matted-row production and local markets in cold climate regions have not been widely tested for adaptation to an annual production cycle. Productivity of seven short-day varieties developed for matted-row and/or annual production was examined in an annual plasticulture system in two consecutive trials in central NY (lat. 42.87° N, long. 76.99° W) harvested in 2013 and 2014. ‘Flavorfest’ demonstrated good performance in Trial 1 with high yield (390 g/plant) and large fruit size (13.9 g mean berry weight). ‘Jewel’ was shown to be well adapted to the annual plasticulture system with consistently high yields (330 and 390 g/plant) that equaled or surpassed other varieties and had moderate fruit size. ‘Chandler’ performed similarly to previous trials conducted in warmer regions with yield (340 g/plant) and fruit size (9.8 g mean berry weight) similar to ‘Jewel’. ‘Clancy’ yielded less but was consistent from year to year. The late season varieties Seneca and Ovation showed marked variability between years, possibly due to drastically different temperatures during flowering and fruit development in Trial 1 compared to Trial 2. High temperatures in Trial 1 likely caused higher early fruit yield, a compressed season and a precipitous decline in fruit size in the later season, thus reducing yield in the late season. Survival after a second dormant period was poor resulting in a small second harvest and reduced fruit size. Overall, the system demonstrated many of the expected benefits but may be more sensitive to weather conditions in the region. While many varieties developed for matted-row production may work well in an annual plasticulture system, not all varieties are equally adapted. Performance of each variety should be determined independently before large scale adoption by growers.

scholarly journals A Novel and Efficient High-Yield Method for Preparing Bacterial Ghosts

Toxins ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 420
Author(s):  
Yi Ma ◽  
Liu Cui ◽  
Meng Wang ◽  
Qiuli Sun ◽  
Kaisheng Liu ◽  
...  
Keyword(s):  
Large Scale ◽  
Expression System ◽  
High Yield ◽  
Wild Type ◽  
High Quality ◽  
Efficient Protection ◽  
Bacterial Ghosts ◽  
Cell Envelopes ◽  
Extracellular Structures ◽  
First Time

Bacterial ghosts (BGs) are empty cell envelopes possessing native extracellular structures without a cytoplasm and genetic materials. BGs are proposed to have significant prospects in biomedical research as vaccines or delivery carriers. The applications of BGs are often limited by inefficient bacterial lysis and a low yield. To solve these problems, we compared the lysis efficiency of the wild-type protein E (EW) from phage ΦX174 and the screened mutant protein E (EM) in the Escherichia coli BL21(DE3) strain. The results show that the lysis efficiency mediated by protein EM was improved. The implementation of the pLysS plasmid allowed nearly 100% lysis efficiency, with a high initial cell density as high as OD600 = 2.0, which was higher compared to the commonly used BG preparation method. The results of Western blot analysis and immunofluorescence indicate that the expression level of protein EM was significantly higher than that of the non-pLysS plasmid. High-quality BGs were observed by SEM and TEM. To verify the applicability of this method in other bacteria, the T7 RNA polymerase expression system was successfully constructed in Salmonella enterica (S. Enterica, SE). A pET vector containing EM and pLysS were introduced to obtain high-quality SE ghosts which could provide efficient protection for humans and animals. This paper describes a novel and commonly used method to produce high-quality BGs on a large scale for the first time.

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