Chromatography affinity resin with photosynthetically-sourced protein A ligand.

Green, photosynthesizing plants can be proficiently used as cost-effective, single-use, fully biodegradable bioreactors for environmentally-friendly production of a variety of valuable recombinant proteins. Being near-infinitely scalable and most energy-efficient in generating biomass, plants represent profoundly valid alternatives to conventionally used stationary fermenters. To validate this, we produced a plastome-engineered tobacco bioreactor line expressing a recombinant variant of the protein A from Staphylococcus aureus, an affinity ligand widely useful in antibody purification processes, reaching accumulation levels up to ~ 250 mg per 1 kg of fresh leaf biomass.

Plant-produced recombinant cytokines IL-37b and IL-38 modulate inflammatory response from stimulated human PBMCs.

Affordable therapeutics are vitally needed for humans worldwide. Plant-based production of recombinant proteins can potentially enhance, back-up, or even substitute for the manufacturing capacity of the conventional, fermenter-based technologies. We plastome-engineered a tobacco cultivar to express high levels of two "plantakines" - recombinant human cytokines, interleukins IL-37b and IL-38, and confirmed their native conformation and folding.

Plastid Transformation of Micro-Tom Tomato with a Hemipteran Double-Stranded RNA Results in RNA Interference in Multiple Insect Species.

Plant-mediated RNA interference (RNAi) holds great promise for insect pest control, as plants can be transformed to produce double-stranded RNA (dsRNA) to selectively down-regulate insect genes essential for survival. For optimum potency, dsRNA can be produced in plant plastids, enabling the accumulation of unprocessed dsRNAs. However, the relative effectiveness of this strategy in inducing an RNAi response in insects using different feeding mechanisms is understudied.

Transplastomic Tomato Plants Expressing Insect-Specific Double-Stranded RNAs: A Protocol Based on Biolistic Transformation.

Expressing insecticidal double-stranded RNA (dsRNA) molecules in plant plastids is a novel approach for in planta production of dsRNA that has enormous potential for developing improved plant-mediated RNA interference (RNAi) strategies for insect pest control. In this chapter, we describe the design of a transformation vector containing an expression cassette which can be used to stably transform plastids of tomato plants for production and accumulation of dsRNA . Such dsRNA can trigger the mechanisms of RNAi in pest insects and selectively suppress the expression of target genes, resulting in lethality.

RNA Interference in the Tobacco Hornworm, , Using Plastid-Encoded Long Double-Stranded RNA.

RNA interference (RNAi) is a promising method for controlling pest insects by silencing the expression of vital insect genes to interfere with development and physiology; however, certain insect Orders are resistant to this process. In this study, we set out to test the ability of -expressed dsRNA synthesized within the plastids to silence gene expression in an insect recalcitrant to RNAi, the lepidopteran species, (tobacco hornworm). Using the () gene as the target, we first evaluated RNAi efficiency of two dsRNA products of different lengths by directly feeding the -synthesized dsRNAs to larvae.

Rapid generation of hypomorphic mutations.

Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation.

The case for plant-made veterinary immunotherapeutics.

The excessive use of antibiotics in food animal production has contributed to resistance in pathogenic bacteria, thereby triggering regulations and consumer demands to limit their use. Alternatives for disease control are therefore required that are cost-effective and compatible with intensive production. While vaccines are widely used and effective, they are available against a minority of animal diseases, and development of novel vaccines and other immunotherapeutics is therefore needed.

Plant-based solutions for veterinary immunotherapeutics and prophylactics.

An alarming increase in emergence of antibiotic resistance among pathogens worldwide has become a serious threat to our ability to treat infectious diseases according to the World Health Organization. Extensive use of antibiotics by livestock producers promotes the spread of new resistant strains, some of zoonotic concern, which increases food-borne illness in humans and causes significant economic burden on healthcare systems. Furthermore, consumer preferences for meat/poultry/fish produced without the use of antibiotics shape today's market demand.

Production and characterization of in planta transiently produced polygalacturanase from Aspergillus niger and its fusions with hydrophobin or ELP tags.

Background: Pectinases play an important role in plant cell wall deconstruction and have potential in diverse industries such as food, wine, animal feed, textile, paper, fuel, and others. The demand for such enzymes is increasing exponentially, as are the efforts to improve their production and to implement their use in several industrial processes. The goal of this study was to examine the potential of producing polygalacturonase I from Aspergillus niger in plants and to investigate the effects of subcellular compartmentalization and protein fusions on its accumulation and activity.

Optimization of transplastomic production of hemicellulases in tobacco: effects of expression cassette configuration and tobacco cultivar used as production platform on recombinant protein yields.

Background: Chloroplast transformation in tobacco has been used extensively to produce recombinant proteins and enzymes. Chloroplast expression cassettes can be designed with different configurations of the cis-acting elements that govern foreign gene expression. With the aim to optimize production of recombinant hemicellulases in transplastomic tobacco, we developed a set of cassettes that incorporate elements known to facilitate protein expression in chloroplasts and examined expression and accumulation of a bacterial xylanase XynA.

Production of a subunit vaccine candidate against porcine post-weaning diarrhea in high-biomass transplastomic tobacco.

Post-weaning diarrhea (PWD) in piglets is a major problem in piggeries worldwide and results in severe economic losses. Infection with Enterotoxigenic Escherichia coli (ETEC) is the key culprit for the PWD disease. F4 fimbriae of ETEC are highly stable proteinaceous polymers, mainly composed of the major structural subunit FaeG, with a capacity to evoke mucosal immune responses, thus demonstrating a potential to act as an oral vaccine against ETEC-induced porcine PWD.

Expressing yeast SAMdc gene confers broad changes in gene expression and alters fatty acid composition in tomato fruit.

Tomato (Solanum lycopersicum) fruits expressing a yeast S-adenosyl methionine decarboxylase (ySAMdc) gene under control of a ripening-induced promoter show altered phytonutrient content and broad changes in gene expression. Genome-wide transcriptional alterations in pericarp tissues of the ySAMdc-expressing fruits are shown. Consistent with the ySAMdc expression pattern from the ripening-induced promoter, very minor transcriptional alterations were detected at the mature green developmental stage.

Microarray analysis and functional tests suggest the involvement of expansins in the early stages of symbiosis of the arbuscular mycorrhizal fungus Glomus intraradices on tomato (Solanum lycopersicum).

Arbuscular mycorrhizal (AM) symbiosis occurs between fungi of the phylum Glomeromycota and most terrestrial plants. However, little is known about the molecular symbiotic signalling between AM fungi (AMFs) and non-leguminous plant species. We sought to further elucidate the molecular events occurring in tomato, a non-leguminous host plant, during the early, pre-symbiotic stage of AM symbiosis, i.

Transcriptional profiling of high pigment-2dg tomato mutant links early fruit plastid biogenesis with its overproduction of phytonutrients.

Phenotypes of the tomato (Solanum lycopersicum) high pigment-2(dg) (hp-2(dg)) and hp-2(j) mutants are caused by lesions in the gene encoding DEETIOLATED1, a negative regulator of light signaling. Homozygous hp-2(dg) and hp-2(j) plants display a plethora of distinctive developmental and metabolic phenotypes in comparison to their normal isogenic counterparts. These mutants are, however, best known for the increased levels of carotenoids, primarily lycopene, and other plastid-accumulating functional metabolites.