scholarly journals Production of Human IFNγ Protein in Nicotiana benthamiana Plant through an Enhanced Expression System Based on Bamboo mosaic Virus

Viruses ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 509 ◽  
Author(s):  
Min-Chao Jiang ◽  
Chung-Chi Hu ◽  
Na-Sheng Lin ◽  
Yau-Heiu Hsu
Keyword(s):  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Expression System ◽  
Therapeutic Protein ◽  
Biologically Active ◽  
Total Soluble Protein ◽  
Expression Vectors ◽  
Human Interferon ◽  
Protein Accumulation ◽  
Expression Systems

Plant-based systems are safe alternatives to the current platforms for the production of biologically active therapeutic proteins. However, plant-based expression systems face certain major challenges, including the relatively low productivity and the generation of target proteins in biologically active forms. The use of plant virus-based expression systems has been shown to enhance yields, but further improvement is still required to lower the production cost. In this study, various strategies were employed to increase the yields of an important therapeutic protein, human interferon gamma (IFNγ), in Nicotiana benthamiana through modifications of expression vectors based on potexviruses. Among these, the vector based on a coat protein (CP)-deficient Bamboo mosaic virus (BaMV), pKB△CHis, was shown to exhibit the highest expression level for the unmodified IFNγ. Truncation of the N-terminal signal peptide of IFN (designated mIFNγ) resulted in a nearly seven-fold increase in yield. Co-expression of a silencing suppressor protein by replacing the coding sequence of BaMV movement protein with that of P19 led to a 40% increase in mIFNγ accumulation. The fusion of endoplasmic reticulum (ER) retention signal with mIFNγ significantly enhanced the accumulation ratio of biologically active dimeric mIFNγ to 87% relative to the non-active monomeric form. The construct pKB19mIFNγER, employing the combination of all the above enhancement strategies, gave the highest level of protein accumulation, up to 119 ± 0.8 μg/g fresh weight, accounting for 2.5% of total soluble protein (TSP) content. These findings advocate the application of the modified BaMV-based vector as a platform for high-level expression of therapeutic protein in N. benthamiana.

scholarly journals The new plant expression system for the development of vaccines against papillomaviruses

2019 ◽  
Vol 484 (4) ◽  
pp. 503-506 ◽  
Author(s):  
R. K. Salyaev ◽  
N. I. Rekoslavskaya ◽  
A. S. Stolbikov
Keyword(s):  
High Risk ◽  
Mosaic Virus ◽  
Soluble Protein ◽  
Tomato Fruit ◽  
Expression System ◽  
Total Soluble Protein ◽  
Coat Proteins ◽  
Gene Encoding ◽  
Genetic Construct ◽  
Antigenic Proteins

To enhance the synthesis of the main antigenic coat proteins L1 of high risk oncogenic papillomaviruses types HPV16, HPV18, HPV31 and HPV45, the sequence of the gene encoding the cucumber mosaic virus replicase (RdRP CMV) was inserted into the genetic construct. This inclusion has made possible to increase highly the production of these antigenic proteins to 25—27 µg per 1 mg of total soluble protein of transformed tomato fruit.

scholarly journals A Method for Rapid Screening, Expression, and Purification of Antimicrobial Peptides

2021 ◽  
Vol 9 (9) ◽  
pp. 1858
Author(s):  
Yingli Zhang ◽  
Zhongchen Li ◽  
Li Li ◽  
Ben Rao ◽  
Lixin Ma ◽  
...  
Keyword(s):  
Antimicrobial Peptides ◽  
Fusion Proteins ◽  
Expression System ◽  
Inhibition Zone ◽  
Eukaryotic Expression ◽  
Biologically Active ◽  
Rapid Screening ◽  
Expression Vectors ◽  
Expression And Purification ◽  
E Coli

In this study, a method for the rapid screening, expression and purification of antimicrobial peptides (AMPs) was developed. AMP genes were fused to a heat-resistant CL7 tag using the SLOPE method, and cloned into Escherichia coli and Pichia pastoris expression vectors. Twenty E. coli and ten P. pastoris expression vectors were constructed. Expression supernatants were heated, heteroproteins were removed, and fusion proteins were purified by nickel affinity (Ni-NTA) chromatography. Fusion proteins were digested on the column using human rhinovirus (HRV) 3C protease, and AMPs were released and further purified. Five AMPs (1, 2, 6, 13, 16) were purified using the E. coli expression system, and one AMP (13) was purified using the P. pastoris expression system. Inhibition zone and minimum inhibitory concentration (MIC) tests confirmed that one P. pastoris¬-derived and two E. coli-derived AMPs have the inhibition activity. The MIC of AMP 13 and 16 from E. coli was 24.2 μM, and the MIC of AMP 13 from P. pastoris was 8.1 μM. The combination of prokaryotic and eukaryotic expression systems expands the universality of the developed method, facilitating screening of a large number of biologically active AMPs, establishing an AMP library, and producing AMPs by industrialised biological methods.

scholarly journals Soluble Expression and Characterization of Biologically Active Bacillus anthracis Protective Antigen in Escherichia coli

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Nagendra Suryanarayana ◽  
Vanlalhmuaka ◽  
Bharti Mankere ◽  
Monika Verma ◽  
Kulanthaivel Thavachelvam ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Expression System ◽  
Lethal Factor ◽  
Biologically Active ◽  
Expression Vectors ◽  
Soluble Form ◽  
Small Scale ◽  
E Coli

Bacillus anthracis secretory protein protective antigen (PA) is primary candidate for subunit vaccine against anthrax. Attempts to obtain large quantity of PA from Escherichia coli expression system often result in the formation of insoluble inclusion bodies. Therefore, it is always better to produce recombinant proteins in a soluble form. In the present study, we have obtained biologically active recombinant PA in small scale E. coli shake culture system using three different expression constructs. The PA gene was cloned in expression vectors bearing trc, T5, and T7 promoters and transformed into their respective E. coli hosts. The growth conditions were optimized to obtain maximum expression of PA in soluble form. The expression construct PA-pET32c in DE3-pLysS E. coli host resulted in a maximum production of soluble PA (15 mg L−1) compared to other combinations. Purified PA was subjected to trypsin digestion and binding assay with lethal factor to confirm the protein’s functionality. Biological activity was confirmed by cytotoxicity assay on J774.1 cells. Balb/c mice were immunized with PA and the immunogenicity was tested by ELISA and toxin neutralization assay. This study highlights the expression of soluble and biologically active recombinant PA in larger quantity using simpler E. coli production platform.

Heterologous expression of synthetic chicken IFN-γ in transgenic tobacco plants

Biologia ◽  
2009 ◽  
Vol 64 (6) ◽  
Author(s):  
Yongjun Wu ◽  
Degang Zhao ◽  
Li Song ◽  
Wenzhao Xu
Keyword(s):  
Transgenic Tobacco ◽  
Tobacco Plant ◽  
Expression System ◽  
Biologically Active ◽  
Total Soluble Protein ◽  
Pcr Analysis ◽  
Therapeutic Effects ◽  
Tobacco Plants ◽  
Tobacco Leaves ◽  
Ifn Γ

AbstractTo develop a plant expression system for the production of the chicken interferon gamma (ChIFN-γ) oral vaccine adjuvant, we investigated whether the ChIFN-γ protein can be expressed in tobacco plants. The coding sequence of the ChIFN-γ gene was optimized by modification of codon usage to that of tobacco plant genes. A synthetic ChIFN-γ gene was inserted into plasmid pSW-IFNG containing the CaMV35S promoter, NOS terminator, GUS (β-glucuronidase) reporter gene and the nptII resistance gene. The synthetic ChIFN-γ gene, along with an endoplasmic reticulum retention signal (SEKDEL) was introduced into tobacco plants via Agrobacterium-mediated transformation. PCR analysis confirmed the presence of the ChIFN-γ gene in transformants. Histochemical GUS assay of the tobacco leaves revealed stable integration of ChIFN-γ into the genome. RT-PCR analysis revealed the presence of ChIFN-γ-specific transcript. Bands of approximately 30 and 40 kDa were detected by Western analysis of transgenic tobacco leaves using an anti-chicken IFN-γ antibody. Furthermore, quantitative ELISA detected ChIFN-γ protein, suggesting that tobacco leaves expressed ChIFN-γ of up to 10 to 20 µg/g fresh leaf weight, or 0.02 to 0.04% of total soluble protein. ChIFN-γ present in tobacco extracts possessed antiviral activity (4.8×105 IU/mg in vitro). The present results indicate that the ChIFN-γ introduced into tobacco plant was correctly transcribed and translated in plant. High-level expression of biologically active ChIFN-γ will allow further studies of oral administration and therapeutic effects of this cytokine.

Seeds as Economical Production Platform for Recombinant Proteins

2020 ◽  
Vol 27 (2) ◽  
pp. 89-104 ◽  
Author(s):  
Muhammad Sarwar Khan ◽  
Faiz Ahmad Joyia ◽  
Ghulam Mustafa
Keyword(s):  
Oral Delivery ◽  
High Protein ◽  
Biologically Active ◽  
Cold Chain ◽  
Protein Accumulation ◽  
Long Term Storage ◽  
Production Platform ◽  
Vaccine Antigens ◽  
Term Storage

: The cost-effective production of high-quality and biologically active recombinant molecules especially proteins is extremely desirable. Seed-based recombinant protein production platforms are considered as superior choice owing to lack of human/animal pathogenic organisms, lack of cold chain requirements for transportation and long-term storage, easy scalability and development of edible biopharmaceuticals in plants with objective to be used in purified or partially processed form is desirable. This review article summarizes the exceptional features of seed-based biopharming and highlights the needs of exploiting it for commercial purposes. Plant seeds offer a perfect production platform for high-value molecules of industrial as well as therapeutic nature owing to lower water contents, high protein storage capacity, weak protease activity and long-term storage ability at ambient temperature. Exploiting extraordinarily high protein accumulation potential, vaccine antigens, antibodies and other therapeutic proteins can be stored without effecting their stability and functionality up to years in seeds. Moreover, ability of direct oral consumption and post-harvest stabilizing effect of seeds offer unique feature of oral delivery of pharmaceutical proteins and vaccine antigens for immunization and disease treatment through mucosal as well as oral route.

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 Genome-Wide Identification and Characterization of Long Noncoding RNAs Involved in Chinese Wheat Mosaic Virus Infection of Nicotiana benthamiana

Biology ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 232
Author(s):  
Weiran Zheng ◽  
Haichao Hu ◽  
Qisen Lu ◽  
Peng Jin ◽  
Linna Cai ◽  
...  
Keyword(s):  
Virus Infection ◽  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Noncoding Rnas ◽  
Long Noncoding Rnas ◽  
Differentially Expressed ◽  
Genome Wide ◽  
Chinese Wheat

Recent studies have shown that a large number of long noncoding RNAs (lncRNAs) can regulate various biological processes in animals and plants. Although lncRNAs have been identified in many plants, they have not been reported in the model plant Nicotiana benthamiana. Particularly, the role of lncRNAs in plant virus infection remains unknown. In this study, we identified lncRNAs in N. benthamiana response to Chinese wheat mosaic virus (CWMV) infection by RNA sequencing. A total of 1175 lncRNAs, including 65 differentially expressed lncRNAs, were identified during CWMV infection. We then analyzed the functions of some of these differentially expressed lncRNAs. Interestingly, one differentially expressed lncRNA, XLOC_006393, was found to participate in CWMV infection as a precursor to microRNAs in N. benthamiana. These results suggest that lncRNAs play an important role in the regulatory network of N. benthamiana in response to CWMV infection.

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