A Combination of Flexible Modified Plant Virus Nanoparticles Enables Additive Effects Resulting in Improved Osteogenesis.

Plant virus nanoparticles (VNPs) genetically engineered to present osteogenic cues provide a promising method for biofunctionalizing hydrogels in bone tissue engineering. Flexible Potato virus X (PVX) nanoparticles substantially enhance the attachment and differentiation of human mesenchymal stem cells (hMSCs) by presenting the RGD motif, hydroxyapatite-binding peptide (HABP), or consecutive polyglutamates (E8) in a concentration-dependent manner. Therefore, it is hypothesized that Tobacco mosaic virus nanoparticles, which present 1.6 times more functional peptides than PVX, will meliorate such an impact. This study hypothesizes that cultivating hMSCs on a surface coated with a combination of two VNPs presenting peptides for either cell attachment or mineralization can achieve additionally enhancing effects on osteogenesis. Calcium minerals deposited by differentiating hMSCs increases two to threefold for this combination, while the Alkaline Phosphatase activity of hMSCs grown on the PVX-RGD/PVX-HABP-coated surface significantly surpasses any other VNP combination. Superior additive effects are observed for the first time by employing a combination of VNPs with varying functionalities. It is found that the flexible VNP geometry plays a more critical role than the concentration of functional peptides. In conclusion, various peptide-presenting plant VNPs exhibit an additive enhancing effect offering significant potential for effectively functionalizing cell-containing hydrogels in bone tissue engineering.

Scaling eukaryotic cell-free protein synthesis achieved with the versatile and high-yielding tobacco BY-2 cell lysate.

Eukaryotic cell-free protein synthesis (CFPS) can accelerate expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and difficulties scaling such systems have prevented their widespread adoption in protein research and manufacturing. Here, we provide detailed demonstrations for the capabilities of a CFPS system derived from Nicotiana tabacum BY-2 cell culture (BY-2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in 48 h, complete with native disulfide bonds and N-glycosylation. An optimized version of the technology is commercialized as ALiCE® and advances in scaling of BYL production methodologies now allow scaling of eukaryotic CFPS reactions. We show linear, lossless scale-up of batch mode protein expression from 100 µL microtiter plates to 10 and 100 mL volumes in Erlenmeyer flasks, culminating in preliminary data from a litre-scale reaction in a rocking-type bioreactor. Together, scaling across a 20,000x range is achieved without impacting product yields. Production of multimeric virus-like particles from the BYL cytosolic fraction were then shown, followed by functional expression of multiple classes of complex, difficult-to-express proteins using the native microsomes of the BYL CFPS. Specifically: a dimeric enzyme; a monoclonal antibody; the SARS-CoV-2 receptor-binding domain; a human growth factor; and a G protein-coupled receptor membrane protein. Functional binding and activity are demonstrated, together with in-depth PTM characterization of purified proteins through disulfide bond and N-glycan analysis. Taken together, BYL is a promising end-to-end R&D to manufacturing platform with the potential to significantly reduce the time-to-market for high value proteins and biologics.

The concept of an agroinfiltration kit for recombinant protein production for educational and commercial use-A journey through a forest of regulatory and legal implications.

Recombinant expression using Agrobacterium-mediated transient transformation (ATT) of plants has developed into a robust and versatile method to rapidly produce proteins. The capability of plants to efficiently synthesize even homo- and hetero-multimeric complex folded proteins featuring disulfide bonds and other post-translational modifications such as N-linked glycosylation makes them superior to most of the established microbial, especially prokaryotic expression hosts. Compared to production in mammalian cell cultures, ATT requires lower skills, simple technical equipment and cheaper media components. Taken together these features make the method optimally suited for R&D applications involving the development and engineering of recombinant proteins for various purposes ranging from vaccine candidates, therapeutic proteins, towards enzymes for different pharmaceutical and technical applications. Despite these advantages the technology is currently not being used outside the community of plant research. The design and realization of a kit containing all the information, instructions and ideally also the material required to perform recombinant protein production using ATT in an educational or commercial context was one of the objectives of the EU-funded Horizon 2020 project Pharma-Factory. While it is pretty straightforward to assemble a comprehensive instruction manual describing the procedure, the clarification of regulatory and legal aspects associated with the provision, dissemination and use of the different materials and organisms required to perform ATT is a complex matter. In this article, we describe the initial concept of an ATT kit for educational as well as research and development (R&D) purposes and the specific regulatory and legal implications associated with the various kit components. We cover aspects including intellectual property rights, freedom-to-operate (FTO), safety regulations for distributing genetically-modified organisms (GMOs), as well as export and import regulations. Our analysis reveals that important components of the ATT kit are freely available for research purposes but not or only with considerable effort for commercial use and distribution. We conclude with a number of considerations and requirements that need to be met in order to successfully disseminate such a kit in the future.

Production of Recombinant Proteins by Agrobacterium-Mediated Transient Expression.

The agroinfiltration of plant tissue is a robust method that allows the rapid and transient expression of recombinant proteins. Using wild-type plants as biomass, agroinfiltration exploits the ability of plants to synthesize even complex multimeric proteins that require oxidative folding and/or post-translational modifications, while avoiding the expensive and time-consuming creation of stably transformed plant lines. Here we describe a generic method for the transient expression of recombinant proteins in Nicotiana benthamiana at the small to medium laboratory scale, including appropriate binary vectors, the design and cloning of expression constructs, the transformation, selection, and cultivation of recombinant Agrobacterium tumefaciens, the infiltration of plants using a syringe or vacuum device, and finally the extraction of recombinant proteins from plant tissues.

Plant-Based Cell-Free Transcription and Translation of Recombinant Proteins.

Plant cell-free lysates contain all the cellular components of the protein biosynthesis machinery, providing an alternative to intact plant cells, tissues, and whole plants for the production of recombinant proteins. Cell-free lysates achieve rapid protein production (within hours or days) and allow the synthesis of proteins that are cytotoxic or unstable in living cells. The open nature of cell-free lysates and their homogeneous and reproducible performance is ideal for protein production, especially for screening applications, allowing the direct addition of nucleic acid templates encoding proteins of interest, as well as other components such as enzyme substrates, chaperones, artificial amino acids, or labeling molecules. Here we describe procedures for the production of recombinant proteins in the ALiCE (Almost Living Cell-free Expression) system, a lysate derived from tobacco cell suspension cultures that can be used to manufacture protein products for molecular and biochemical analysis as well as applications in the pharmaceutical industry.

Rapid production of SaCas9 in plant-based cell-free lysate for activity testing.

Cas9 nucleases have become the most versatile tool for genome editing projects in a broad range of organisms. The recombinant production of Cas9 nuclease is desirable for in vitro activity assays or the preparation of ribonucleoproteins (RNPs) for DNA-free genome editing approaches. For the rapid production of Cas9, we explored the use of a recently established cell-free lysate from tobacco (Nicotiana tabacum L.) BY-2 cells. Using this system, the 130-kDa Cas9 nuclease from Staphylococcus aureus (SaCas9) was produced and subsequently purified via affinity chromatography. The purified apoenzyme was supplemented with 10 different sgRNAs, and the nuclease activity was confirmed by the linearization of plasmid DNA containing cloned DNA target sequences.

Targeted mutagenesis in Nicotiana tabacum ADF gene using shockwave-mediated ribonucleoprotein delivery increases osmotic stress tolerance.

DNA-free genome editing involves the direct introduction of ribonucleoprotein (RNP) complexes into cells, but this strategy has rarely been successful in plants. In the present study, we describe a new technique for the introduction of RNPs into plant cells involving the generation of cavitation bubbles using a pulsed laser. The resulting shockwave achieves the efficient transfection of walled cells in tissue explants by creating transient membrane pores. RNP-containing cells were rapidly identified by fluorescence microscopy, followed by regeneration and the screening of mutant plants by high-resolution melt analysis. We used this technique in Nicotiana tabacum to target the endogenous phytoene desaturase (PDS) and actin depolymerizing factor (ADF) genes. Genome-edited plants were produced with an efficiency of 35.2% for PDS and 16.5% for ADF. Further we evaluated the physiological, cellular and molecular effects of ADF mutations in T2 mutant plants under drought and salinity stress. The results suggest that ADF acts as a key regulator of osmotic stress tolerance in plants.

Selection and characterization of two monoclonal antibodies specific for the Aspergillus flavus major antigenic cell wall protein Aflmp1.

Aspergillus flavus is a major fungal pathogen of plants and an opportunistic pathogen of humans. In addition to the direct impact of infection, it produces immunosuppressive and carcinogenic aflatoxins. The early detection of A. flavus is therefore necessary to diagnose and monitor fungal infection, to prevent aflatoxin contamination of food and feed, and for effective antifungal therapy. Aspergillus-specific monoclonal antibodies (mAbs) are promising as diagnostic and therapeutic reagents for the tracking and treatment of Aspergillus infections, respectively. However, A. flavus has a complex cell wall composition and dynamic morphology, hindering the discovery of mAbs with well-characterized targets. Here we describe the generation and detailed characterization of mAb5.52 (IgG2aκ) and mAb17.15 (IgG1κ), which bind specifically to the highly immunogenic cell wall antigen A. flavus mannoprotein 1 (Aflmp1). Both mAbs were generated using hybridoma technology following the immunization of mice with a recombinant truncated version of Aflmp1 (ExD, including the homologous CR4 domain) produced in bacteria. We show that mAb5.52 and mAb17.15 bind specifically to A. flavus and A. parasiticus cell wall fragments (CWFs), with no cross-reaction to CWFs from other fungal pathogens. Immunofluorescence microscopy revealed that both mAbs bind to the surface of Aspergillus hyphae and that mAb17.15 also binds to spores. The epitope for both mAbs is localized within the CR4 region of the Aflmp1 protein. These Aspergillus-specific mAbs may be useful for the early detection of fungal infection in food/feed crops, for serodiagnosis in patients with invasive aspergillosis caused by A. flavus infection and for the development of antibody-expressing disease-resistant crops.

Impact of nicotine pathway downregulation on polyamine biosynthesis and leaf ripening in tobacco.

Traditional breeding and molecular approaches have been used to develop tobacco varieties with reduced nicotine and secondary alkaloid levels. However, available low-alkaloid tobacco varieties have impaired leaf quality likely due to the metabolic consequences of nicotine biosynthesis downregulation. Recently, we found evidence that the unbalanced crosstalk between nicotine and polyamine pathways is involved in impaired leaf ripening of a low-alkaloid (LA) Burley 21 line having a mutation at the Nic1 and Nic2 loci, key biosynthetic regulators of nicotine biosynthesis. Since the Nic1 and Nic2 loci are comprised of several genes, all phenotypic changes seen in LA Burley 21 could be due to a mixture of genetics-based responses. Here, we investigated the commercial burley variety TN90 LC and its transgenic versions with only one downregulated gene, either putrescine methyl transferase (PMT-RNAi) or PR50-protein (PR50-RNAi). Nicotine levels of cured lamina of TN90 LC, TN90 PMT-RNAi and TN90 PR50-RNAi, were 70.5 ± 3.8, 2.4 ± 0.5, and 6.0 ± 1.1 mg/g dry weight, respectively. Low-alkaloid transgenic lines showed delayed leaf maturation and impaired leaf quality. We analyzed polyamine contents and ripening markers in wild-type TN90 control plants (WT) and the two transgenic lines. The ripening markers revealed that the PMT-RNAi line showed the most pronounced impaired leaf maturation phenotype at harvest, characterized by higher chlorophyll (19%) and glucose (173%) contents and more leaf mesophyll cells per area (25%), while the ripening markers revealed that maturation of PR50-RNAi plants was intermediate between PMT-RNAi and WT lines. Comparative polyamine analyses showed an increase in free and conjugated polyamines in roots of both transgenic lines, this being most pronounced in the PMT-RNAi plants. For PMT-RNAi plants, there were further perturbations of polyamine content in the leaves, which mirrored the general phenotype, as PR50-RNAi transgenic plants looked more similar to the WT than PMT-RNAi transgenic plants. Activity of ornithine decarboxylase, the enzyme that catalyzes the committing step of polyamine biosynthesis, was significantly higher in roots and mature leaves of PMT-RNAi plants in comparison to WT, while there was no increase observed for arginine decarboxylase. Treatment of both transgenic lines with polyamine biosynthesis inhibitors decreased the polyamine content and ameliorated the phenotype, confirming the intricate interplay of polyamine and nicotine biosynthesis in tobacco and the influence of this interplay on leaf ripening.

Plant molecular farming for the production of valuable proteins - Critical evaluation of achievements and future challenges.

Recombinant proteins play an important role in many areas of our lives. For example, recombinant enzymes are used in the food and chemical industries and as high-quality proteins for research, diagnostic and therapeutic applications. The production of recombinant proteins is still dominated by expression systems based on microbes and mammalian cells, although the manufacturing of recombinant proteins in plants - known as molecular farming - has been promoted as an alternative, cost-efficient strategy for three decades. Several molecular farming products have reached the market, but the number of success stories has been limited by industrial inertia driven by perceptions of low productivity, the high cost of downstream processing, and regulatory hurdles that create barriers to translation. Here, we discuss the technical and economic factors required for the successful commercialization of molecular farming, and consider potential future directions to enable the broader application of production platforms based on plants.

Plant-Derived Cell-Free Biofactories for the Production of Secondary Metabolites.

Cell-free expression systems enable the production of proteins and metabolites within a few hours or days. Removing the cellular context while maintaining the protein biosynthesis apparatus provides an open system that allows metabolic pathways to be installed and optimized by expressing different numbers and combinations of enzymes. This facilitates the synthesis of secondary metabolites that are difficult to produce in cell-based systems because they are toxic to the host cell or immediately converted into downstream products. Recently, we developed a cell-free lysate derived from tobacco BY-2 cell suspension cultures for the production of recombinant proteins. This system is remarkably productive, achieving yields of up to 3 mg/mL in a one-pot in vitro transcription-translation reaction and contains highly active energy and cofactor regeneration pathways. Here, we demonstrate for the first time that the BY-2 cell-free lysate also allows the efficient production of several classes of secondary metabolites. As case studies, we synthesized lycopene, indigoidine, betanin, and betaxanthins, which are useful in the food, cosmetic, textile, and pharmaceutical industries. Production was achieved by the co-expression of up to three metabolic enzymes. For all four products, we achieved medium to high yields. However, the yield of betanin (555 µg/mL) was outstanding, exceeding the level reported in yeast cells by a factor of more than 30. Our results show that the BY-2 cell-free lysate is suitable not only for the verification and optimization of metabolic pathways, but also for the efficient production of small to medium quantities of secondary metabolites.

Saturation mutagenesis to improve the degradation of azo dyes by versatile peroxidase and application in form of VP-coated yeast cell walls.

Azo dyes are toxic and carcinogenic synthetic pigments that accumulate as pollutants in aquatic bodies near textile factories. The pigments are structurally diverse, and bioremediation is mostly limited to single dye compounds or related groups. Versatile peroxidase (VP) from Pleurotus eryngii is a heme-containing peroxidase with a broad substrate spectrum that can break down many structurally distinct pollutants, including azo dyes. The utilization of this enzyme could be facilitated by engineering to modify its catalytic activity and substrate range. We used saturation mutagenesis to alter two amino acids in the catalytic tryptophan environment of VP (V160 and A260). Library screening with three azo dyes revealed that these two positions had a significant influence on substrate specificity. We were able to isolate and sequence VP variants with up to 16-fold higher catalytic efficiency for different azo dyes. The same approach could be used to select for VP variants that catalyze the degradation of many other types of pollutants. To allow multiple cycles of dye degradation, we immobilized VP on the surface of yeast cells and used washed cell wall fragments after lysis. VP embedded in the cell wall retained ∼70 % of its initial activity after 10 cycles of dye degradation each lasting 12 h, making this platform ideal for the bioremediation of environments contaminated with azo dyes.

Targeted insertion of large DNA sequences by homology-directed repair or non-homologous end joining in engineered tobacco BY-2 cells using designed zinc finger nucleases.

Targeted integration of recombinant DNA fragments into plant genomes by DNA double-strand break (DSB) repair mechanisms has become a powerful tool for precision engineering of crops. However, many targeting platforms require the screening of many transgenic events to identify a low number of targeted events among many more random insertion events. We developed an engineered transgene integration platform (ETIP) that uses incomplete marker genes at the insertion site to enable rapid phenotypic screening and recovery of targeted events upon functional reconstitution of the marker genes. The two marker genes, encoding neomycin phosphotransferase II (nptII) and Discosoma sp. red fluorescent protein (DsRed) enable event selection on kanamycin-containing selective medium and subsequent screening for red fluorescent clones. The ETIP design allows targeted integration of donor DNA molecules either by homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated mechanisms. Targeted donor DNA integration is facilitated by zinc finger nucleases (ZFN). The ETIP cassette was introduced into Nicotiana tabacum BY-2 suspension cells to generate target cell lines containing a single copy locus of the transgene construct. The utility of the ETIP platform has been demonstrated by targeting DNA constructs containing up to 25-kb payload. The success rate for clean targeted DNA integration was up to 21% for HDR and up to 41% for NHEJ based on the total number of calli analyzed by next-generation sequencing (NGS). The rapid generation of targeted events with large DNA constructs expands the utility of the nuclease-mediated gene addition platform both for academia and the commercial sector.

The Integration of Algal Carbon Concentration Mechanism Components into Tobacco Chloroplasts Increases Photosynthetic Efficiency and Biomass.

Increasing the productivity of crops is a major challenge in agricultural research. Given that photosynthetic carbon assimilation is necessary for plant growth, enhancing the efficiency of photosynthesis is one strategy to boost agricultural productivity. The authors attempted to increase the photosynthetic efficiency and biomass of tobacco plants by expressing individual components of the Chlamydomonas reinhardtii carbon concentration mechanism (CCM) and integrating them into the chloroplast. Independent transgenic varieties are generated accumulating the carbonic anhydrase CAH3 in the thylakoid lumen or the bicarbonate transporter LCIA in the inner chloroplast membrane. Independent homozygous transgenic lines showed enhanced CO2 uptake rates (up to 15%), increased photosystem II efficiency (by up to 18%), and chlorophyll content (up to 19%). Transgenic lines produced more shoot biomass than wild-type and azygous controls, and accumulated more carbohydrate and amino acids, reflecting the higher rate of photosynthetic CO2 fixation. These data demonstrate that individual algal CCM components can be integrated into C3 plants to increase biomass, suggesting that transgenic lines combining multiple CCM components could further increase the productivity and yield of C3 crops.

Plant-derived chimeric antibodies inhibit the invasion of human fibroblasts by Toxoplasma gondii.

The parasite Toxoplasma gondii causes an opportunistic infection, that is, particularly severe in immunocompromised patients, infants, and neonates. Current antiparasitic drugs are teratogenic and cause hypersensitivity-based toxic side effects especially during prolonged treatment. Furthermore, the recent emergence of drug-resistant toxoplasmosis has reduced the therapeutic impact of such drugs. In an effort to develop recombinant antibodies as a therapeutic alternative, a panel of affinity-matured, T. gondii tachyzoite-specific single-chain variable fragment (scFv) antibodies was selected by phage display and bioinformatic analysis. Further affinity optimization was attempted by introducing point mutations at hotspots within light chain complementarity-determining region 2. This strategy yielded four mutated scFv sequences and a parental scFv that were used to produce five mouse-human chimeric IgGs in Nicotiana benthamiana plants, with yields of 33-72 mg/kg of plant tissue. Immunological analysis confirmed the specific binding of these plant-derived antibodies to T. gondii tachyzoites, and in vitro efficacy was demonstrated by their ability to inhibit the invasion of human fibroblasts and impair parasite infectivity. These novel recombinant antibodies could therefore be suitable for the development of plant-derived immunotherapeutic interventions against toxoplasmosis.

Aspergillus-specific antibodies - Targets and applications.

Aspergillus is a fungal genus comprising several hundred species, many of which can damage the health of plants, animals and humans by direct infection and/or due to the production of toxic secondary metabolites known as mycotoxins. Aspergillus-specific antibodies have been generated against polypeptides, polysaccharides and secondary metabolites found in the cell wall or secretions, and these can be used to detect and monitor infections or to quantify mycotoxin contamination in food and feed. However, most Aspergillus-specific antibodies are generated against heterogeneous antigen preparations and the specific target remains unknown. Target identification is important because this can help to characterize fungal morphology, confirm host penetration by opportunistic pathogens, detect specific disease-related biomarkers, identify new candidate targets for antifungal drug design, and qualify antibodies for diagnostic and therapeutic applications. In this review, we discuss how antibodies are raised against heterogeneous Aspergillus antigen preparations and how they can be characterized, focusing on strategies to identify their specific antigens and epitopes. We also discuss the therapeutic, diagnostic and biotechnological applications of Aspergillus-specific antibodies.

Polyamines delay leaf maturation in low-alkaloid tobacco varieties.

The development of low-alkaloid (LA) tobacco varieties is an important target in the tobacco breeding industry. However, LA Burley 21 plants, in which the Nic1 and Nic2 loci controlling nicotine biosynthesis are deleted, are characterized by impaired leaf maturation that leads to poor leaf quality before and after curing. Polyamines are involved in key developmental, physiological, and metabolic processes in plants, and act as anti-senescence and anti-ripening regulators. We investigated the role of polyamines in tobacco leaf maturation by analyzing the free and conjugated polyamine fractions in the leaves and roots of four Burley 21 varieties: NA (normal alkaloid levels, wild-type control), HI (high intermediates, nic2 -), LI (low intermediates, nic1 -), and LA (nic1 - nic2 -). The pool of conjugated polyamines increased with plant age in the roots and leaves of all four varieties, but the levels of free and conjugated putrescine and spermidine were higher in the LI and LA plants than NA controls. The increase in the polyamine content correlated with delayed maturation and senescence, i.e., LA plants with the highest polyamine levels showed the most severe impaired leaf maturation phenotype, characterized by higher chlorophyll content and more mesophyll cells per unit leaf area. Treatment of LA plants with inhibitors of polyamine biosynthesis and/or the growth regulator Ethephon® reduced accumulation of polyamines, achieving a partial amelioration of the LA phenotype. Our data show that the regulation of polyamine homeostasis is strongly disrupted in LA plants, and that free and conjugated polyamines contribute to the observed impairment of leaf maturation.

Animal component-free Agrobacterium tumefaciens cultivation media for better GMP-compliance increases biomass yield and pharmaceutical protein expression in Nicotiana benthamiana.

Transient expression systems allow the rapid production of recombinant proteins in plants. Such systems can be scaled up to several hundred kilograms of biomass, making them suitable for the production of pharmaceutical proteins required at short notice, such as emergency vaccines. However, large-scale transient expression requires the production of recombinant Agrobacterium tumefaciens strains with the capacity for efficient gene transfer to plant cells. The complex media often used for the cultivation of this species typically include animal-derived ingredients that can contain human pathogens, thus conflicting with the requirements of good manufacturing practice (GMP). We replaced all the animal-derived components in yeast extract broth (YEB) cultivation medium with soybean peptone, and then used a design-of-experiments approach to optimize the medium composition, increasing the biomass yield while maintaining high levels of transient expression in subsequent infiltration experiments. The resulting plant peptone Agrobacterium medium (PAM) achieved a two-fold increase in OD600 compared to YEB medium during a 4-L batch fermentation lasting 18 h. Furthermore, the yields of the monoclonal antibody 2G12 and the fluorescent protein DsRed were maintained when the cells were cultivated in PAM rather than YEB. We have thus demonstrated a simple, efficient and scalable method for medium optimization that reduces process time and costs. The final optimized medium for the cultivation of A. tumefaciens completely lacks animal-derived components, thus facilitating the GMP-compliant large-scale transient expression of recombinant proteins in plants.

Structured plant metabolomics for the simultaneous exploration of multiple factors.

Multiple factors act simultaneously on plants to establish complex interaction networks involving nutrients, elicitors and metabolites. Metabolomics offers a better understanding of complex biological systems, but evaluating the simultaneous impact of different parameters on metabolic pathways that have many components is a challenging task. We therefore developed a novel approach that combines experimental design, untargeted metabolic profiling based on multiple chromatography systems and ionization modes, and multiblock data analysis, facilitating the systematic analysis of metabolic changes in plants caused by different factors acting at the same time. Using this method, target geraniol compounds produced in transgenic tobacco cell cultures were grouped into clusters based on their response to different factors. We hypothesized that our novel approach may provide more robust data for process optimization in plant cell cultures producing any target secondary metabolite, based on the simultaneous exploration of multiple factors rather than varying one factor each time. The suitability of our approach was verified by confirming several previously reported examples of elicitor-metabolite crosstalk. However, unravelling all factor-metabolite networks remains challenging because it requires the identification of all biochemically significant metabolites in the metabolomics dataset.

A Plant-Based Transient Expression System for the Rapid Production of Malaria Vaccine Candidates.

There are currently no vaccines that provide sterile immunity against malaria. Various proteins from different stages of the Plasmodium falciparum life cycle have been evaluated as vaccine candidates, but none of them have fulfilled expectations. Therefore, combinations of key antigens from different stages of the parasites life cycle may be essential for the development of efficacious malaria vaccines. Following the identification of promising antigens using bioinformatics, proteomics, and/or immunological approaches, it is necessary to express, purify, and characterize these proteins and explore the potential of fusion constructs combining different antigens or antigen domains before committing to expensive and time-consuming clinical development. Here, using malaria vaccine candidates as an example, we describe how Agrobacterium tumefaciens-based transient expression in plants can be combined with a modular and flexible cloning strategy as a robust and versatile tool for the rapid production of candidate antigens during research and development.