Transient Expression in Cytoplasm and Apoplast of Rotavirus VP6 Protein Fused to Anti-DEC205 Antibody in Nicotiana benthamiana and Nicotiana sylvestris.
Ontology highlight
ABSTRACT:
SUBMITTER: Castillo-Esparza JF
PROVIDER: S-EPMC8214057 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:To optimize the expression, extraction and purification of plant-derived tetrameric recombinant human butyrylcholinesterase (prBChE), we describe the development and use of plant viral amplicon-based gene expression system; Tobacco Mosaic Virus (TMV) RNA-based overexpression vector (TRBO) to express enzymatically active FLAG-tagged plant made recombinant butyrylcholinesterase (rBChE) in Nicotiana benthamiana leaves using transient agroinfiltration. Two gene expression cassettes were designed to express the recombinant protein in either the ER or to the apoplastic compartment. Leaf homogenization was used to isolate ER-retained recombinant butyrylcholinesterase (prBChE-ER) while apoplast-targeted rBChE was isolated by either leaf homogenization (prBChE) or vacuum-extraction of apoplastic wash fluid (prBChE-AWF). rBChE from apoplast wash fluid had a higher specific activity but lower enzyme yield than leaf homogenate. To optimize the isolation and purification of total recombinant protein from leaf homogenates, an acidic extraction buffer was used. The acidic extraction buffer yielded >95% enzymatically active tetrameric rBChE as verified by Coomassie stained and native gel electrophoresis. Furthermore, when compared to human butyrylcholinesterase, the prBChE was found to be similar in terms of tetramerization and enzyme kinetics. The N-linked glycan profile of purified prBChE-ER was found to be mostly high mannose structures while the N-linked glycans on prBChE-AWF were primarily complex. The glycan profile of the prBChE leaf homogenates showed a mixture of high mannose, complex and paucimannose type N-glycans. These findings demonstrate the ability of plants to produce rBChE that is enzymatically active and whose oligomeric state is comparable to mammalian butyrylcholinesterase. The process of plant made rBChE tetramerization and strategies for improving its pharmacokinetics properties are also discussed.
| S-EPMC4909763 | biostudies-literature
Project description:BackgroundCharacterization of plant terpene synthases is typically done by production of recombinant enzymes in Escherichia coli. This is often difficult due to solubility and codon usage issues. Furthermore, plant terpene synthases which are targeted to the plastids, such as diterpene synthases, have to be shortened in a more or less empirical approach to improve expression. We report here an optimized Agrobacterium-mediated transient expression assay in Nicotiana benthamiana for plant diterpene synthase expression and product analysis.ResultsAgrobacterium-mediated transient expression of plant diterpene synthases in N. benthamiana led to the accumulation of diterpenes within 3 days of infiltration and with a maximum at 5 days. Over 50% of the products were exported onto the leaf surface, thus considerably facilitating the analysis by reducing the complexity of the extracts. The robustness of the method was tested by expressing three different plant enzymes, cembratrien-ol synthase from Nicotiana sylvestris, casbene synthase from Ricinus communis and levopimaradiene synthase from Gingko biloba. Furthermore, co-expression of a 1-deoxy-D-xylulose-5-phosphate synthase from tomato and a geranylgeranyl diphosphate synthase from tobacco led to a 3.5-fold increase in the amount of cembratrien-ol produced, with maximum yields reaching 2500 ng/cm2.ConclusionWith this optimized method for diterpene synthase expression and product analysis, a single infiltrated leaf of N. benthamiana would be sufficient to produce quantities required for the structure elucidation of unknown diterpenes. The method will also be of general use for gene function discovery, pathway reconstitution and metabolic engineering of diterpenoid biosynthesis in plants.
| S-EPMC3878842 | biostudies-literature
Project description:Human rotaviruses are the main cause of severe gastroenteritis in children and are responsible for over 500 000 deaths annually. There are two live rotavirus vaccines currently available, one based on human rotavirus serotype G1P[8], and the other a G1-G4 P[8] pentavalent vaccine. However, the recent emergence of the G9 and other novel rotavirus serotypes in Africa and Asia has prompted fears that current vaccines might not be fully effective against these new varieties.We report an effort to develop an affordable candidate rotavirus vaccine against the new emerging G9P[6] (RVA/Human-wt/ZAF/GR10924/1999/G9P[6]) strain. The vaccine is based on virus-like particles which are both highly immunogenic and safe. The vaccine candidate was produced in Nicotiana benthamiana by transient expression, as plants allow rapid production of antigens at lower costs, without the risk of contamination by animal pathogens. Western blot analysis of plant extracts confirmed the successful expression of two rotavirus capsid proteins, VP2 and VP6. These proteins assembled into VLPs resembling native rotavirus particles when analysed by transmission electron microscopy (TEM). Expression of the rotavirus glycoprotein VP7 and the spike protein VP4 was also tried. However, VP7 expression caused plant wilting during the course of the time trial and expression could never be detected for either protein. We therefore created three fusion proteins adding the antigenic part of VP4 (VP8*) to VP6 in an attempt to produce more appropriately immunogenic particles. Fusion protein expression in tobacco plants was detected by western blot using anti-VP6 and anti-VP4 antibodies, but no regular particles were observed by TEM, even when co-expressed with VP2.Our results suggest that the rotavirus proteins produced in N. benthamiana are candidates for a subunit vaccine specifically for the G9P[6] rotavirus strain. This could be more effective in developing countries, thereby possibly providing a higher overall efficacy for the existing vaccines. The production of rotavirus proteins in plants would probably result in lower manufacturing costs, making it more affordable for developing countries. Further investigation is required to evaluate the immunogenic potential of the VLPs and fusion proteins created in this study.
| S-EPMC4667453 | biostudies-literature
Project description:Horseradish peroxidase (HRP) is a commercially important reagent enzyme used in molecular biology and in the diagnostic product industry. It is typically purified from the roots of the horseradish (Armoracia rusticana); however, this crop is only available seasonally, yields are variable and often low, and the product is a mixture of isoenzymes. Engineering high-level expression in transiently transformed tobacco may offer a solution to these problems. In this study, a synthetic Nicotiana benthamiana codon-adapted full-length HRP isoenzyme gene as well as C-terminally truncated and both N- and C-terminally truncated versions of the HRP C gene were synthesized, and their expression in N. benthamiana was evaluated using an Agrobacterium tumefaciens-mediated transient expression system. The influence on HRP C expression levels of co-infiltration with a silencing suppressor (NSs) construct was also evaluated. Highest HRP C levels were consistently obtained using either the full length or C-terminally truncated HRP C constructs. HRP C purification by ion exchange chromatography gave an overall yield of 54% with a Reinheitszahl value of >3 and a specific activity of 458 U/mg. The high level of HRP C production in N. benthamiana in just five days offers an alternative, viable, and scalable system for production of this commercially significant enzyme.
| S-EPMC5796064 | biostudies-literature
Project description:Flaviviruses are a threat to public health and can cause major disease outbreaks. Tick-borne encephalitis (TBE) is caused by a flavivirus, and it is one of the most important causes of viral encephalitis in Europe and is on the rise in Sweden. As there is no antiviral treatment available, vaccination remains the best protective measure against TBE. Currently available TBE vaccines are based on formalin-inactivated virus produced in cell culture. These vaccines must be delivered by intramuscular injection, have a burdensome immunization schedule, and may exhibit vaccine failure in certain populations. This project aimed to develop an edible TBE vaccine to trigger a stronger immune response through oral delivery of viral antigens to mucosal surfaces. We demonstrated successful expression and post-translational processing of flavivirus structural proteins which then self-assembled to form virus-like particles in Nicotiana benthamiana. We performed oral toxicity tests in mice using various plant species as potential bioreactors and evaluated the immunogenicity of the resulting edible vaccine candidate. Mice immunized with the edible vaccine candidate did not survive challenge with TBE virus. Interestingly, immunization of female mice with a commercial TBE vaccine can protect their offspring against TBE virus infection.
| S-EPMC9610170 | biostudies-literature
Project description:BackgroundAgroinfiltration is a simple and effective method of delivering transgenes into plant cells for the rapid production of recombinant proteins and has become the preferred transient expression platform to manufacture biologics in plants. Despite its popularity, few studies have sought to improve the efficiency of agroinfiltration to further increase protein yields. This study aimed to increase agroinfiltration-based transient gene expression in Nicotiana benthamiana by improving all levels of transgenesis.ResultsUsing the benchmark pEAQ-HT deconstructed virus vector system and the GUS reporter enzyme, physical, chemical, and molecular features were independently assessed for their ability to enhance Agrobacterium-mediated transformation and improve protein production capacities. Optimal Agrobacterium strain, cell culture density and co-cultivation time for maximal transient GUS (β-glucuronidase) expression were established. The effects of chemical additives in the liquid infiltration media were investigated and acetosyringone (500 μM), the antioxidant lipoic acid (5 μM), and a surfactant Pluronic F-68 (0.002%) were all shown to significantly increase transgene expression. Gene products known to suppress post-transcriptional gene silencing, activate cell cycle progression and confer stress tolerance were also assessed by co-expression. A simple 37 °C heat shock to plants, 1-2 days post infiltration, was shown to dramatically increase GUS reporter levels. By combining the most effective features, a dual vector delivery system was developed that provided approximately 3.5-fold higher levels of absolute GUS protein compared to the pEAQ-HT platform.ConclusionsIn this paper, different strategies were assessed and optimised with the aim of increasing plant-made protein capacities in Nicotiana benthamiana using agroinfiltration. Chemical additives, heat shock and the co-expression of genes known to suppress stress and gene silencing or stimulate cell cycle progression were all proven to increase agroinfiltration-based transient gene expression. By combining the most effective of these elements a novel expression platform was developed capable of producing plant-made protein at a significantly higher level than a benchmark hyper-expression system.
| S-EPMC6109318 | biostudies-literature
Project description:Correct subcellular targeting is crucial for protein function. Protein location can be visualized in vivo by fusion to a fluorescent protein (FP). Nevertheless, despite intense engineering efforts, most FPs are dim or completely quenched at low pH (<6). This is particularly problematic for the study of proteins targeted to acidic compartments such as vacuoles (pH ~ 3-6) or plant cell walls (pH ~ 3.5-8.3). Plant cell walls play important roles (e.g. structural/protective role, control of growth/morphogenesis), are diverse in structure and function, and are highly dynamic (e.g. during cell growth, in response to biotic/abiotic stresses). To study and engineer plant cell walls, it is therefore critical to identify robust tools which can be used to locate proteins expressed in the apoplast. Here we used a transient expression assay in Nicotiana benthamiana leaves to test a range of FPs in vivo, and determined which ones retained strong fluorescence in the acidic environment of the apoplast. We selected 10 fluorescent proteins with a range of in vitro properties; two historical FPs and eight FPs with in vitro properties suggesting lower pH sensitivity or improved brightness, some of which had never been tested in plants prior to our study. We targeted each FP to the cytosol or the apoplast and compared the fluorescence in both compartments, before testing the in vivo pH sensitivity of FPs across a pH 8-4 gradient. Our results suggest that mTurquoise2, mNeonGreen, and mCherry are suited to tracking proteins in the apoplast under dynamic pH conditions. These fluorescent proteins may also be useful in other acidic compartments such as vacuoles.
| S-EPMC6508812 | biostudies-literature
Project description:Plant secondary metabolites have applications for the food, biofuel, and pharmaceutical industries. Recent advances in pathway elucidation and host expression systems now allow metabolic engineering of plant metabolic pathways to produce "new-to-nature" derivatives with novel biological activities, thereby amplifying the range of industrial uses for plant metabolites. Here we use a transient expression system in the model plant Nicotiana benthamiana to reconstitute the two-step plant-derived biosynthetic pathway for auxin (indole acetic acid) to achieve accumulation up to 500 ng/g fresh mass (FM). By expressing these plant-derived enzymes in combination with either bacterial halogenases and alternative substrates, we can produce both natural and new-to-nature halogenated auxin derivatives up to 990 ng/g FM. Proteins from the auxin synthesis pathway, tryptophan aminotransferases (TARs) and flavin-dependent monooxygenases (YUCs), could be transiently expressed in combination with four separate bacterial halogenases to generate halogenated auxin derivatives. Brominated auxin derivatives could also be observed after infiltration of the transfected N. benthamiana with potassium bromide and the halogenases. Finally, the production of additional auxin derivatives could also be achieved by co-infiltration of TAR and YUC genes with various tryptophan analogs. Given the emerging importance of transient expression in N. benthamiana for industrial scale protein and product expression, this work provides insight into the capacity of N. benthamiana to interface bacterial genes and synthetic substrates to produce novel halogenated metabolites.
| S-EPMC7714751 | biostudies-literature
Project description:Candidatus Liberibacter asiaticus "Las" is a phloem-limited bacterial plant pathogen, and the most prevalent species of Liberibacter associated with citrus huanglongbing (HLB), a devastating disease of citrus worldwide. Although, the complete sequence of the Las genome provides the basis for studying functional genomics of Las and molecular mechanisms of Las-plant interactions, the functional characterization of Las effectors remains a slow process since remains to be cultured. Like other plant pathogens, Las may deliver effector proteins into host cells and modulate a variety of host cellular functions for their infection progression. In this study, we identified 16 putative Las effectors via bioinformatics, and transiently expressed them in Nicotiana benthamiana. Diverse subcellular localization with different shapes and aggregation patterns of the effector candidates were revealed by UV- microscopy after transient expression in leaf tissue. Intriguingly, one of the 16 candidates, Las5315mp (mature protein), was localized in the chloroplast and induced cell death at 3 days post inoculation (dpi) in N. benthamiana. Moreover, Las5315mp induced strong callose deposition in plant cells. This study provides new insights into the localizations and potential roles of these Las effectors in planta.
| S-EPMC4933711 | biostudies-literature
Project description:Bluetongue virus (BTV) causes severe disease in domestic and wild ruminants, and has recently caused several outbreaks in Europe. Current vaccines include live-attenuated and inactivated viruses; while these are effective, there is risk of reversion to virulence by mutation or reassortment with wild type viruses. Subunit or virus-like particle (VLP) vaccines are safer options: VLP vaccines produced in insect cells by expression of the four BTV capsid proteins are protective against challenge; however, this is a costly production method. We investigated production of BTV VLPs in plants via Agrobacterium-mediated transient expression, an inexpensive production system very well suited to developing country use. Leaves infiltrated with recombinant pEAQ-HT vectors separately encoding the four BTV-8 capsid proteins produced more proteins than recombinant pTRA vectors. Plant expression using the pEAQ-HT vector resulted in both BTV-8 core-like particles (CLPs) and VLPs; differentially controlling the concentration of infiltrated bacteria significantly influenced yield of the VLPs. In situ localisation of assembled particles was investigated by using transmission electron microscopy (TEM) and it was shown that a mixed population of core-like particles (CLPs, consisting of VP3 and VP7) and VLPs were present as paracrystalline arrays in the cytoplasm of plant cells co-expressing all four capsid proteins.
| S-EPMC5360979 | biostudies-literature