scholarly journals Expression of novel fusion antiviral proteins Ricin A Chain-Pokeweed Antiviral Proteins (RTA-PAPs) in Escherichia coli and their inhibition of protein synthesis and of hepatitis B virus in vitro

2018 ◽  
Author(s):  
Yasser Hassan ◽  
Sherry Ogg ◽  
Hui Ge
Keyword(s):  
Protein Synthesis ◽  
Expression System ◽  
Therapeutic Index ◽  
Antiviral Protein ◽  
E Coli ◽  
Protein Isoform ◽  
Antiviral Proteins ◽  
A Chain

AbstractRicin A chain (RTA) and Pokeweed antiviral proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential antiviral value of novel fusion proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-Pokeweed antiviral protein isoform 1 from seeds (RTA-PAPS1) was produced in E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and protein synthesis inhibitory activity assayed by comparison to the production of a control protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. Results showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded protein was bioactive with 50% protein synthesis inhibitory concentration (IC50) of 0.06nM (3.63ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with a half-maximal response concentration value (EC50) of 0.03nM (1.82ng/ml) and a therapeutic index of >21818. The fusion protein was further optimized using in silico tools, produced in E. coli in vivo expression system, purified by three-step process from soluble lysate and protein synthesis inhibition activity assayed. Results showed that the optimized protein RTA mutant-Pokeweed antiviral protein isoform 1 from leaves (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function activity on protein synthesis inhibition with an IC50 of 0.03nM (1.82ng/ml). Collectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant antiviral activity against HBV in vitro with a high therapeutic index and, thus, meriting further development as potential antiviral agents against chronic HBV infection.

scholarly journals Expression of Pokeweed Antiviral Protein Isoform S1 (PAP-S1) and of Ricin-A-Chain/PAP-S1 novel fusion protein (RTA/PAP-S1) in Escherichia coli and their comparative inhibition of protein synthesis in vitro

2017 ◽  
Author(s):  
Yasser Hassan ◽  
Sherry Ogg
Keyword(s):  
Protein Synthesis ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Antiviral Protein ◽  
Developmental Potential ◽  
E Coli ◽  
Protein Isoform ◽  
A Chain

AbstractFusion protein therapeutics engineering is advancing to meet the need for novel medicine. Herein, we further characterize the development of novel RTA & PAP-S1 antiviral fusion proteins. In brief, RTA/PAP-S1 and PAP-S1/RTA fusion proteins were produced in both cell free and E. coli in vivo expression systems, purified by His-tag affinity chromatography, and protein synthesis inhibitory activity assayed by comparison to the production of a control protein, CalmL3. Results showed that the RTA/PAP-S1 fusion protein is amenable to standardized production and purification and has both increased potency and less toxicity compared to either RTA or PAP-S1 alone. Thus, this research highlights the developmental potential of novel fusion proteins with reduced cytotoxic risk and increased potency.

scholarly journals Expression of Pokeweed Antiviral Protein Isoform S1 (PAP-S1) and of Ricin-A-Chain/PAP-S1 Novel Fusion Protein (RTA/PAP-S1) in Escherichia coli and Their Comparative Inhibition of Protein Synthesis in Vitro

2017 ◽  
Author(s):  
Yasser Hassan ◽  
Sherry Ogg
Keyword(s):  
Protein Synthesis ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Antiviral Protein ◽  
Developmental Potential ◽  
E Coli ◽  
Protein Isoform ◽  
A Chain

Fusion protein therapeutics engineering is advancing to meet the need for novel medicine. Herein, we further characterize the development of novel RTA & PAP-S1 antiviral fusion proteins. In brief, RTA/PAP-S1 and PAP-S1/RTA fusion proteins were produced in both cell free and E. coli in vivo expression systems, purified by His-tag affinity chromatography, and protein synthesis inhibitory activity assayed by comparison to the production of a control protein, CalmL3. Results showed that the RTA/PAP-S1 fusion protein is amenable to standardized production and purification and has both increased potency and less toxicity compared to either RTA or PAP-S1 alone. Thus, this research highlights the developmental potential of novel fusion proteins with reduced cytotoxic risk and increased potency.

scholarly journals A Neutralizing Antibody to the A Chain of Abrin Inhibits Abrin Toxicity both In Vitro and In Vivo

2008 ◽  
Vol 15 (5) ◽  
pp. 737-743 ◽  
Author(s):  
Kalpana Surendranath ◽  
Anjali A. Karande
Keyword(s):  
Monoclonal Antibody ◽  
Protein Synthesis ◽  
Neutralizing Antibody ◽  
Type Ii ◽  
Inhibit Protein Synthesis ◽  
Ribosome Inactivating Proteins ◽  
A Chain ◽  
Lethal Doses

ABSTRACT Plant ribosome-inactivating proteins (RIPs) are RNA N-glycosidases that inhibit protein synthesis in cells. Abrin, a type II RIP, is an AB type toxin, which is one of the most lethal types of toxin known. The B chain facilitates the entry of the molecule into the cell, whereas the A chain exerts the toxic effect. We have generated hybridomas secreting antibodies of the immunoglobulin G class specific to the recombinant A chain of abrin. One monoclonal antibody, namely, D6F10, rescued cells from abrin toxicity. Importantly, the antibody also protected mice from lethal doses of the toxin. The neutralizing effect of the antibody was shown to be due to interference with abrin attachment to the cell surface.

Compartmentalization of an all-E. coli Cell-Free Expression System for the Construction of a Minimal Cell

2016 ◽  
Vol 22 (2) ◽  
pp. 185-195 ◽  
Author(s):  
Filippo Caschera ◽  
Vincent Noireaux
Keyword(s):  
Protein Synthesis ◽  
Expression System ◽  
Coli Cell ◽  
Free Expression ◽  
Genetic Circuit ◽  
Translation System ◽  
Minimal Cell ◽  
E Coli ◽  
Synthetic Cell

Cell-free expression is a technology used to synthesize minimal biological cells from natural molecular components. We have developed a versatile and powerful all-E. coli cell-free transcription–translation system energized by a robust metabolism, with the far objective of constructing a synthetic cell capable of self-reproduction. Inorganic phosphate (iP), a byproduct of protein synthesis, is recycled through polysugar catabolism to regenerate ATP (adenosine triphosphate) and thus supports long-lived and highly efficient protein synthesis in vitro. This cell-free TX-TL system is encapsulated into cell-sized unilamellar liposomes to express synthetic DNA programs. In this work, we study the compartmentalization of cell-free TX-TL reactions, one of the aspects of minimal cell module integration. We analyze the signals of various liposome populations by fluorescence microscopy for one and for two reporter genes, and for an inducible genetic circuit. We show that small nutrient molecules and proteins are encapsulated uniformly in the liposomes with small fluctuations. However, cell-free expression displays large fluctuations in signals among the same population, which are due to heterogeneous encapsulation of the DNA template. Consequently, the correlations of gene expression with the compartment dimension are difficult to predict accurately. Larger vesicles can have either low or high protein yields.

scholarly journals Production and Evaluation of an Avian IgY Immunotoxin against CD133+ for Treatment of Carcinogenic Stem Cells in Malignant Glioma: IgY Immunotoxin for the Treatment of Glioblastoma

2019 ◽  
Vol 2019 ◽  
pp. 1-15 ◽  
Author(s):  
Elda-Georgina Chavez-Cortez ◽  
Gustavo Vargas Felix ◽  
Edgar Rangel López ◽  
Julio Sotelo ◽  
Carlos Martínez-Canseco ◽  
...  
Keyword(s):  
Stem Cells ◽  
Malignant Glioma ◽  
Potential Therapeutic Target ◽  
E Coli ◽  
Novel Approach ◽  
Original Goal ◽  
Design Production ◽  
A Chain

Background. Glioblastoma is the most common malignant tumor of Central Nervous System. Despite the research in therapeutics, the prognosis is dismal. Malignant glioma stem cells (MGSCs) are a major cause of treatment failure and increasing tumor recurrence. In general, cancer stem cells (CSCs) express prominin-1 (CD133), considered as a potential therapeutic target. In this study, we produced an avian immunotoxin directed against the subpopulation of CD133+ CSCs within a malignant glioma. We used the avian IgY because it has various advantages as increased affinity to mammal antigens and inexpensive obtention of large amounts of specific antibodies (approximately 1 mg/per egg). The design, production, purification and use of IgY anti CD133 immunotoxin constitute an original goal of this research.Methods. The immunodominant peptide of CD133 was designed to immunize hens; also, the extracellular domain of CD133 was cloned to probe the IgY antibodies. In parallel, a recombinant abrin A chain was produced inE. coliin order to join it to the Fc domain of the anti-CD133 IgY to conform the immunotoxin. This anti-CD133 IgY anti-tumor immunotoxin was testedin vitroandin vivo. Results. The cytotoxicity of the immunotoxinin vitroshowed that IgY-abrin immunotoxin reduced 55% cell viability. After subcutaneous MGSCs implantation, the animals treated intraperitoneally or intratumorally with the IgY-abrin immunotoxin showed more than 50% decrease of tumor volume.Conclusion. Results showed that the IgY-abrin immunotoxin had cytotoxic activity against CD133+ MGSCs and provides a novel approach for the immunotherapy of glioblastoma.

scholarly journals Incompatibility of DFHBI based fluorescent RNA aptamers with particular commercial cell-free expression systems

2021 ◽  
Author(s):  
Alexander J Speakman ◽  
Katherine E Dunn
Keyword(s):  
Expression System ◽  
In Vitro Transcription ◽  
Rna Aptamers ◽  
Free Expression ◽  
Reaction Products ◽  
Expression Systems ◽  
E Coli ◽  
Cell Free Expression System

Fluorescent RNA aptamers are an increasingly used tool for quantifying transcription and for visualising RNA interactions, both in vitro and in vivo. However when tested in the commercially available, E. coli extract based Expressway™ cell-free expression system, no fluorescence is detected. The same experimental setup is shown to successfully produce fluorescent RNA aptamers when tested in another buffer designed for in vitro transcription, and RNA purification of the Expressway™ reaction products show that transcription does occur, but does not result in a fluorescent product. In this paper we demonstrate the incompatibility of a narrow selection of RNA aptamers in one particular cell-free expression system, and consider that similar issues may arise with other cell-free expression systems, RNA aptamers, and their corresponding fluorophores.

scholarly journals Mechanism of action of oxazolidinones: effects of linezolid and eperezolid on translation reactions.

1997 ◽  
Vol 41 (10) ◽  
pp. 2132-2136 ◽  
Author(s):  
D L Shinabarger ◽  
K R Marotti ◽  
R W Murray ◽  
A H Lin ◽  
E P Melchior ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Pathogenic Bacteria ◽  
Coli Strain ◽  
In Vitro Translation ◽  
E Coli ◽  
Initiation Phase ◽  
New Class ◽  
Bacterial Translation

The oxazolidinones are a new class of synthetic antibiotics with good activity against gram-positive pathogenic bacteria. Experiments with a susceptible Escherichia coli strain, UC6782, demonstrated that in vivo protein synthesis was inhibited by both eperezolid (formerly U-100592) and linezolid (formerly U-100766). Both linezolid and eperezolid were potent inhibitors of cell-free transcription-translation in E. coli, exhibiting 50% inhibitory concentrations (IC50s) of 1.8 and 2.5 microM, respectively. The ability to demonstrate inhibition of in vitro translation directed by phage MS2 RNA was greatly dependent upon the amount of RNA added to the assay. For eperezolid, 128 microg of RNA per ml produced an IC50 of 50 microM whereas a concentration of 32 microg/ml yielded an IC50 of 20 microM. Investigating lower RNA template concentrations in linezolid inhibition experiments revealed that 32 and 8 microg of MS2 phage RNA per ml produced IC50s of 24 and 15 microM, respectively. This phenomenon was shared by the translation initiation inhibitor kasugamycin but not by streptomycin. Neither oxazolidinone inhibited the formation of N-formylmethionyl-tRNA, elongation, or termination reactions of bacterial translation. The oxazolidinones appear to inhibit bacterial translation at the initiation phase of protein synthesis.

scholarly journals Screening and Identification of Novel cGAS Homologues Using a Combination of in Vitro and In Vivo Protein Synthesis

2019 ◽  
Vol 21 (1) ◽  
pp. 105 ◽  
Author(s):  
Jascha Rolf ◽  
Regine Siedentop ◽  
Stephan Lütz ◽  
Katrin Rosenthal
Keyword(s):  
Protein Synthesis ◽  
Expression System ◽  
Bald Eagle ◽  
Functional Improvement ◽  
Mole Rat ◽  
Screening And Identification ◽  
First Time ◽  
Cell Free Protein Synthesis

The cyclic GMP-AMP synthase (cGAS) catalyzes the synthesis of the multifunctional second messenger, cGAMP, in metazoans. Although numerous cGAS homologues are predicted in protein databases, the catalytic activity towards cGAMP synthesis has been proven for only four of them. Therefore, we selected five novel and yet uncharacterized cGAS homologues, which cover a broad range in the field of vertebrates. Cell-free protein synthesis (CFPS) was used for a pre-screening to investigate if the cGAS genes originating from higher organisms can be efficiently expressed in a bacterial expression system. As all tested cGAS variants were expressible, enzymes were synthesized in vivo to supply higher amounts for a subsequent in vitro activity assay. The assays were carried out with purified enzymes and revealed vast differences in the activity of the homologues. For the first time, the cGAS homologues from the Przewalski’s horse, naked mole-rat, bald eagle, and zebrafish were proven to catalyze the synthesis of cGAMP. The extension of the list of described cGAS variants enables the acquisition of further knowledge about the structural and molecular mechanism of cGAS, potentially leading to functional improvement of the enzyme.

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