scholarly journals Transient expression and purification of β-caryophyllene synthase in Nicotiana benthamiana to produce β-caryophyllene in vitro

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8904
Author(s):  
Saraladevi Muthusamy ◽  
Ramesh R. Vetukuri ◽  
Anneli Lundgren ◽  
Suresh Ganji ◽  
Li-Hua Zhu ◽  
...  
Keyword(s):  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Artemisia Annua ◽  
Molecular Farming ◽  
Expression System ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gas Chromatography Mass Spectrometry ◽  
Fresh Weight

The sesquiterpene β-caryophyllene is an ubiquitous component in many plants that has commercially been used as an aroma in cosmetics and perfumes. Recent studies have shown its potential use as a therapeutic agent and biofuel. Currently, β-caryophyllene is isolated from large amounts of plant material. Molecular farming based on the Nicotiana benthamiana transient expression system may be used for a more sustainable production of β-caryophyllene. In this study, a full-length cDNA of a new duplicated β-caryophyllene synthase from Artemisia annua (AaCPS1) was isolated and functionally characterized. In order to produce β-caryophyllene in vitro, the AaCPS1 was cloned into a plant viral-based vector pEAQ-HT. Subsequently, the plasmid was transferred into the Agrobacterium and agroinfiltrated into N. benthamiana leaves. The AaCPS1 expression was analyzed by quantitative PCR at different time points after agroinfiltration. The highest level of transcripts was observed at 9 days post infiltration (dpi). The AaCPS1 protein was extracted from the leaves at 9 dpi and purified by cobalt–nitrilotriacetate (Co-NTA) affinity chromatography using histidine tag with a yield of 89 mg kg−1 fresh weight of leaves. The protein expression of AaCPS1 was also confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses. AaCPS1 protein uses farnesyl diphosphate (FPP) as a substrate to produce β-caryophyllene. Product identification and determination of the activity of purified AaCPS1 were done by gas chromatography–mass spectrometry (GC–MS). GC–MS results revealed that the AaCPS1 produced maximum 26.5 ± 1 mg of β-caryophyllene per kilogram fresh weight of leaves after assaying with FPP for 6 h. Using AaCPS1 as a proof of concept, we demonstrate that N. benthamiana can be considered as an expression system for production of plant proteins that catalyze the formation of valuable chemicals for industrial applications.

scholarly journals Long-Term Potato Virus X (PVX)-Based Transient Expression of Recombinant GFP Protein in Nicotiana benthamiana Culture In Vitro

Plants ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2187
Author(s):  
Yana Sindarovska ◽  
Mykola Kuchuk
Keyword(s):  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Molecular Farming ◽  
Transient Gene Expression ◽  
Leaf Explants ◽  
Potato Virus X ◽  
Culture In Vitro ◽  
High Efficient

Plant molecular farming has a great potential to produce valuable proteins. Transient expression technology provides high yields of recombinant proteins in greenhouse-grown plants, but every plant must be artificially agroinfiltrated, and open greenhouse systems are less controlled. Here, we propose to propagate agrobacteria-free plants with high-efficient long-term self-replicated transient gene expression in a well-controlled closed in vitro system. Nicotiana benthamiana plant tissue culture in vitro, with transient expression of recombinant GFP, was obtained through shoot induction from leaf explants infected by a PVX-based vector. The transient expression occurs in new tissues and regenerants due to the natural systemic distribution of viral RNA carrying the target gene. Gene silencing was delayed in plants grown in vitro, and GFP was detected in plants for five to six months. Agrobacteria-free, GFP-expressing plants can be micropropagated in vitro (avoiding an agroinfiltration step), “rejuvenated” through regeneration (maintaining culture for years), or transferred in soil. The mean GFP in the regenerants was 18% of the total soluble proteins (TSP) (0.52 mg/g of fresh leaf weight (FW). The highest value reached 47% TSP (2 mg/g FW). This study proposes a new method for recombinant protein production combining the advantages of transient expression technology and closed cultural systems.

scholarly journals Transient expression of GFP in plant tissue culture in vitro using viral-based module system

1970 ◽  
pp. 198-201
Author(s):  
O. M. Kishchenko ◽  
A. A. Peterson ◽  
M. Yu. Vasylenko ◽  
M. V. Kuchuk
Keyword(s):  
Tissue Culture ◽  
Transient Expression ◽  
Plant Tissue Culture ◽  
Plant Tissue ◽  
Expression System ◽  
Polyacrylamide Gel Electrophoresis ◽  
Plant Production ◽  
Culture In Vitro ◽  
Module System

Aim. Agrobacterium-mediated transient expression using viral-based vectors is one of the most effective method for recombinant protein production in plant. Transient expression of GFP using viral-based module system was studied in plant tissue culture in vitro. Methods. Regenerated shoots and callus clones of Nicotiana benthamiana were agroinfiltrated with viral-based module system. Protein extracts from GFP-positive tissues were resolved by non-reducing polyacrylamide gel electrophoresis. GFP from gel was eluted and GFP fluorescence was measured by fluorescence spectrometry with excitation filter at 395 nm and emission filter at 510 nm. Results. Regenerated shoots and callus clones lines accumulated GFP at 242.0±0.7 and 221.6±4.1 μg per g fresh tissue, respectively. The obtained level of transient expression is comparable with other plant production systems in early stage development. Conclusions. The developed technique shows promise for production of therapeutic proteins and antigens in the short term (14–16 days) by transient expression system in plant tissue culture in vitro. Keywords: viral-based module system, transient expression, recombinant proteins, plant tissue culture, GFP.

scholarly journals Functional Characterization of Pembrolizumab Produced in Nicotiana benthamiana Using a Rapid Transient Expression System

2021 ◽  
Vol 12 ◽  
Author(s):  
Tanapati Phakham ◽  
Christine Joy I. Bulaon ◽  
Narach Khorattanakulchai ◽  
Balamurugan Shanmugaraj ◽  
Supranee Buranapraditkun ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Mammalian Cells ◽  
Expression System ◽  
Sodium Dodecyl ◽  
Production Costs ◽  
Plant Production ◽  
Production Platform

The striking innovation and clinical success of immune checkpoint inhibitors (ICIs) have undoubtedly contributed to a breakthrough in cancer immunotherapy. Generally, ICIs produced in mammalian cells requires high investment, production costs, and involves time consuming procedures. Recently, the plants are considered as an emerging protein production platform due to its cost-effectiveness and rapidity for the production of recombinant biopharmaceuticals. This study explored the potential of plant-based system to produce an anti-human PD-1 monoclonal antibody (mAb), Pembrolizumab, in Nicotiana benthamiana. The transient expression of this mAb in wild-type N. benthamiana accumulated up to 344.12 ± 98.23 μg/g fresh leaf weight after 4 days of agroinfiltration. The physicochemical and functional characteristics of plant-produced Pembrolizumab were compared to mammalian cell-produced commercial Pembrolizumab (Keytruda®). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis results demonstrated that the plant-produced Pembrolizumab has the expected molecular weight and is comparable with the Keytruda®. Structural characterization also confirmed that both antibodies have no protein aggregation and similar secondary and tertiary structures. Furthermore, the plant-produced Pembrolizumab displayed no differences in its binding efficacy to PD-1 protein and inhibitory activity between programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) interaction with the Keytruda®. In vitro efficacy for T cell activation demonstrated that the plant-produced Pembrolizumab could induce IL-2 and IFN-γ production. Hence, this proof-of-concept study showed that the plant-production platform can be utilized for the rapid production of functional mAbs for immunotherapy.

scholarly journals Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 524
Author(s):  
Bingqi Wu ◽  
Zhiting Chen ◽  
Xiaohui Xu ◽  
Ronghua Chen ◽  
Siwei Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Functional Characterization ◽  
Transient Gene Expression ◽  
High Mobility ◽  
Gene Expression System

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

scholarly journals Preliminary characterization of the transcriptional and translational products of the Saccharomyces cerevisiae cell division cycle gene CDC28.

1982 ◽  
Vol 2 (4) ◽  
pp. 412-425 ◽  
Author(s):  
S I Reed ◽  
J Ferguson ◽  
J C Groppe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
In Vitro Mutagenesis ◽  
Coding Region ◽  
Loop Analysis ◽  
Partial Digestion ◽  
Polyadenylic Acid

The CDC28 gene was subcloned from a plasmid containing a 6.5-kilobase-pair segment of Saccharomyces cerevisiae DNA YRp7(CDC28-3) by partial digestion with Sau3A and insertion of the resulting fragments into the BamHI sites of YRp7 and pRC1. Recombinant plasmids were obtained containing inserts of 4.4 and 3.1 kilobase pairs which were capable of complementing a cdc28(ts) mutation. R-loop analysis indicated that each yeast insert contained two RNA coding regions of about 0.8 and 1.0 kilobase pairs, respectively. In vitro mutagenesis experiments suggested that the smaller coding region corresponded to the CDC28 gene. When cellular polyadenylic acid-containing RNA, separated by agarose gel electrophoresis after denaturation with glyoxal and transferred to nitrocellulose membrane, was reacted with labeled DNA from the smaller coding region, and RNA species of about 1 kilobase in length was detected. Presumably, the discrepancy in size between the R-loop and electrophoretic determinations is due to a segment of polyadenylic acid which is excluded from the R-loops. By using hybridization of the histone H2B mRNAs to an appropriate probe as a previously determined standards, it was possible to estimate the number of CDC28 mRNA copies per haploid cell as between 6 and 12 molecules. Hybrid release translation performed on the CDC29 mRNA directed the synthesis of a polypeptide of 27,000 daltons, as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This polypeptide was not synthesized when mRNA prepared from a cdc28 nonsense mutant was translated in a parallel fashion. However, if the RNA from a cell containing the CDC28 gene on a plasmid maintained at a high copy number was translated, the amount of in vitro product was amplified fivefold.

C25, an essential RNA polymerase III subunit related to the RNA polymerase II subunit RPB7

1994 ◽  
Vol 14 (9) ◽  
pp. 6164-6170
Author(s):  
P P Sadhale ◽  
N A Woychik
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rna Polymerase Iii ◽  
Sodium Dodecyl ◽  
Polymerase Iii ◽  
Conditional Mutant ◽  
5.8S Rrna ◽  
Cell Growth And Viability

We identified a partially sequenced Saccharomyces cerevisiae gene which encodes a protein related to the S. cerevisiae RNA polymerase II subunit, RPB7. Several lines of evidence suggest that this related gene, YKL1, encodes the RNA polymerase III subunit C25. C25, like RPB7, is present in submolar ratios, easily dissociates from the enzyme, is essential for cell growth and viability, but is not required in certain transcription assays in vitro. YKL1 has ABF-1 and PAC upstream sequences often present in RNA polymerase subunit genes. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility of the YKL1 gene product is equivalent to that of the RNA polymerase III subunit C25. Finally, a C25 conditional mutant grown at the nonpermissive temperature synthesizes tRNA at reduced rates relative to 5.8S rRNA, a hallmark of all characterized RNA polymerase III mutants.

scholarly journals Phosphorylation of synaptic-membrane proteins from ox cerebral cortex in vitro. Preparation of fractions enriched in phosphorylated proteins by using extraction with detergents and urea, and gel filtration

1977 ◽  
Vol 163 (2) ◽  
pp. 369-378 ◽  
Author(s):  
P R Dunkley ◽  
H Holmes ◽  
R Rodnight
Keyword(s):  
Cerebral Cortex ◽  
Cyclic Amp ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Synaptic Membrane ◽  
Hydroxyl Groups ◽  
Phosphorylated Proteins

Synaptic-membrane fragments from ox cerebral cortex contain basal and cyclic AMP-stimulated protein kinase(s) that transfer 32P from [gamma-32P]ATP to hydroxyl groups of serine and threonine residues in membrane-protein substrates. In the present work, labelled membrane fragments were partitioned into soluble and insoluble fractions with Triton X-100, Nonidet P. 40, sodium deoxycholate and urea, and the distribution of 32P-labelled protein in the fractions was determined by polyacrylamide-gel electrophoresis and radioautography. A high percentage of phosphorylated protein sustrates remained insoluble, including those whose phosphorylation was most highly stimulated by cyclic AMP. Whole membrane fragments and samples prepared by detergent extraction were fractionated on Sepharose 6B columns in the presence of low concentrations of sodium dodecyl sulphate and pooled fractions were analysed by polyacrylamide-gel electrophoresis and radioautography. Phosphorylated proteins were fractionated on the basis of their molecular weight, but homogeneous protein was not obtained. The results are discussed in relation to the techniques used and the results obtained in other laboratories.

scholarly journals Isolation and Characterization of Two Proteins fromMoraxella catarrhalis That Bear a Common Epitope

1998 ◽  
Vol 66 (9) ◽  
pp. 4374-4381 ◽  
Author(s):  
John C. McMichael ◽  
Michael J. Fiske ◽  
Ross A. Fredenburg ◽  
Deb N. Chakravarti ◽  
Karl R. VanDerMeid ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Bacterial Attachment ◽  
Vaccine Candidates ◽  
Isolation And Characterization ◽  
Sds Page

ABSTRACT The UspA1 and UspA2 proteins of Moraxella catarrhalisare potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350,000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100°C. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.

scholarly journals Artemisia Naphta: A novel oil extract for sensitive and acne prone skin

2021 ◽  
Vol 5 (1) ◽  
pp. 022-029
Author(s):  
Tao Kan ◽  
Guo Lili ◽  
Fernandez Jose ◽  
Webb Corey ◽  
Liu Junfeng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Artemisia Annua ◽  
Bioactive Molecules ◽  
Gas Chromatography Mass Spectrometry ◽  
Rt Pcr ◽  
Potential Benefits ◽  
Oil Extract ◽  
Skin Conditions ◽  
Pro Inflammatory Cytokines

Background: The plant Artemisia annua has been used in traditional Chinese medicine for many years. Rich in bioactive molecules, the A. annua plant is used to extract the anti-malaria compound artemisinin (< 1%), which results in most of the plant being unutilized. One byproduct of artemisinin extraction is artemisia naphtha (AN), which has yet to be studied extensively. Aims: Study the activity of a novel AN oil extract against microbes, pro-inflammatory cytokines, and dermatological endpoints that are key for eczema and acne pathogenesis to determine if an effective A. annua extract for these skin conditions can be developed. Methods: Gas chromatography-mass spectrometry was performed to determine the composition of AN oil. P. acnes, S. aureus, M. furfur, and C. albicans were cultured to determine minimal inhibitory concentration. in vitro studies utilizing keratinocytes and macrophages were treated with AN oil and gene expression measured by quantitative RT-PCR. A 13-subject clinical trial was performed with 1% AN oil Gel to assess its potential benefits for sensitive and acne prone skin. Results: AN oil upregulates filaggrin gene expression and possesses antimicrobial and anti-inflammatory activity inhibiting LPS, S. aureus and "Th2 induced" pro-inflammatory mediator release (IL-6, IL-8 and TSLP). Clinical assessment of 1% AN Gel shows it reduces acne blemishes and the appearance of redness. Conclusion: Previously an underutilized and unpurified byproduct, AN is now the source to develop the first topical AN oil for cosmetic use with an activity profile that suggests it is effective for those with sensitive and/or acne prone skin.

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