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. To investigate this, we first tested an in vitro-synthesized 189 bp dsRNA matching a highly conserved region of the v-ATPaseA gene from cotton mealybug (Phenacoccus solenopsis) on three insect species from two different orders that use leaf-chewing, lacerate-and-flush, or sap-sucking mechanisms to feed, and showed that the dsRNA significantly down-regulated the target gene. We then developed transplastomic Micro-tom tomato plants to produce the dsRNA in plant plastids and showed that the dsRNA is produced in leaf, flower, green fruit, red fruit, and roots, with the highest dsRNA levels found in the leaf. The plastid-produced dsRNA induced a significant gene down-regulation in insects using leaf-chewing and lacerate-and-flush feeding mechanisms, while sap-sucking insects were unaffected. Our results suggest that plastid-produced dsRNA can be used to control leaf-chewing and lacerate-and-flush feeding insects, but may not be useful for sap-sucking insects.

Protein Storage Vacuoles Originate from Remodeled Preexisting Vacuoles in Arabidopsis thaliana.

Protein storage vacuoles (PSV) are the main repository of protein in dicotyledonous seeds, but little is known about the origins of these transient organelles. PSV are hypothesized to either arise de novo or originate from the preexisting embryonic vacuole (EV) during seed maturation. Here, we tested these hypotheses by studying PSV formation in Arabidopsis (Arabidopsis thaliana) embryos at different stages of seed maturation and recapitulated this process in Arabidopsis leaves reprogrammed to an embryogenic fate by inducing expression of the LEAFY COTYLEDON2 transcription factor. Confocal and immunoelectron microscopy indicated that both storage proteins and tonoplast proteins typical of PSV were delivered to the preexisting EV in embryos or to the lytic vacuole in reprogrammed leaf cells. In addition, sectioning through embryos at several developmental stages using serial block face scanning electron microscopy revealed the 3D architecture of forming PSV. Our results indicate that the preexisting EV is reprogrammed to become a PSV in Arabidopsis.

A hyper-thermostable α-amylase from Pyrococcus furiosus accumulates in Nicotiana tabacum as functional aggregates.

BACKGROUND: Alpha amylase hydrolyzes α-bonds of polysaccharides such as starch and produces malto-oligosaccharides. Its starch saccharification applications make it an essential enzyme in the textile, food and brewing industries. Commercially available α-amylase is mostly produced from Bacillus or Aspergillus. A hyper-thermostable and Ca 2++ independent α-amylase from Pyrococcus furiosus (PFA) expressed in E.coli forms insoluble inclusion bodies and thus is not feasible for industrial applications. RESULTS: We expressed PFA in Nicotiana tabacum and found that plant-produced PFA forms functional aggregates with an accumulation level up to 3.4 g/kg FW (fresh weight) in field conditions. The aggregates are functional without requiring refolding and therefore have potential to be applied as homogenized plant tissue without extraction or purification. PFA can also be extracted from plant tissue upon dissolution in a mild reducing buffer containing SDS. Like the enzyme produced in P. furiosus and in E. coli, plant produced PFA preserves hyper-thermophilicity and hyper-thermostability and has a long shelf life when stored in lyophilized leaf tissue. With tobacco's large biomass and high yield, hyper-thermostable α-amylase was produced at a scale of 42 kg per hectare. CONCLUSIONS: Tobacco may be a suitable bioreactor for industrial production of active hyperthermostable alpha amylase.

Coating Nanoparticles with Plant-Produced Transferrin-Hydrophobin Fusion Protein Enhances Their Uptake in Cancer Cells.

The encapsulation of drugs to nanoparticles may offer a solution for targeted delivery. Here, we set out to engineer a self-assembling targeting ligand by combining the functional properties of human transferrin and fungal hydrophobins in a single fusion protein. We showed that human transferrin can be expressed in Nicotiana benthamiana plants as a fusion with Trichoderma reesei hydrophobins HFBI, HFBII, or HFBIV. Transferrin-HFBIV was further expressed in tobacco BY-2 suspension cells. Both partners of the fusion protein retained their functionality; the hydrophobin moiety enabled migration to a surfactant phase in an aqueous two-phase system, and the transferrin moiety was able to reversibly bind iron. Coating porous silicon nanoparticles with the fusion protein resulted in uptake of the nanoparticles in human cancer cells. This study provides a proof-of-concept for the functionalization of hydrophobin coatings with transferrin as a targeting ligand.

High accumulation in tobacco seeds of hemagglutinin antigen from avian (H5N1) influenza.

Tobacco seeds can be used as a cost effective system for production of recombinant vaccines. Avian influenza is an important respiratory pathogen that causes a high degree of mortality and becomes a serious threat for the poultry industry. A safe vaccine against avian flu produced at low cost could help to prevent future outbreaks. We have genetically engineered tobacco plants to express extracellular domain of hemagglutinin protein from H5N1 avian influenza virus as an inexpensive alternative for production purposes. Two regulatory sequences of seed storage protein genes from Phaseolus vulgaris L. were used to direct the expression, yielding 3.0 mg of the viral antigen per g of seeds. The production and stability of seed-produced recombinant HA protein was characterized by different molecular techniques. The aqueous extract of tobacco seed proteins was used for subcutaneous immunization of chickens, which developed antibodies that inhibited the agglutination of erythrocytes after the second application of the antigen. The feasibility of using tobacco seeds as a vaccine carrier is discussed.

Protein body formation in leaves of Nicotiana benthamiana: a concentration-dependent mechanism influenced by the presence of fusion tags.

Protein bodies (PBs) are endoplasmic reticulum (ER) derived organelles originally found in seeds whose function is to accumulate seed storage proteins. It has been shown that PB formation is not limited to seeds and green fluorescent protein (GFP) fused to either elastin-like polypeptide (ELP) or hydrophobin (HFBI) fusion tags induce the formation of PBs in leaves of N. benthamiana. In this study, we compared the ELP- and HFBI-induced PBs and showed that ELP-induced PBs are larger than HFBI-induced PBs. The size of ELP- and HFBI-induced PBs increased over time along with the accumulation levels of their fused protein. Our results show that PB formation is a concentration-dependent mechanism in which proteins accumulating at levels higher than 0.2% of total soluble protein are capable of inducing PBs in vivo. Our results show that the presence of fusion tags is not necessary for the formation of PBs, but affects the distribution pattern and size of PBs. This was confirmed by PBs induced by fluorescent proteins as well as fungal xylanases. We noticed that in the process of PB formation, secretory and ER-resident molecules are passively sequestered into the lumen of PBs. We propose to use this property of PBs as a tool to increase the accumulation levels of erythropoietin and human interleukin-10 by co-expression with PB-inducing proteins.

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. RESULTS: Polygalacturonase I from Aspergillus niger (AnPGI) was transiently produced in Nicotiana benthamiana by targeting it to five different cellular compartments: apoplast, endoplasmic reticulum (ER), vacuole, chloroplast and cytosol. Accumulation levels of 2.5%, 3.0%, and 1.9% of total soluble protein (TSP) were observed in the apoplast, ER, and vacuole, respectively, and specific activity was significantly higher in vacuole-targeted AnPGI compared to the same enzyme targeted to the ER or apoplast. No accumulation was found for AnPGI when targeted to the chloroplast or cytosol. Analysis of AnPGI fused with elastin-like polypeptide (ELP) revealed a significant increase in the protein accumulation level, especially when targeted to the vacuole where the protein doubles its accumulation to 3.6% of TSP, while the hydrophobin (HFBI) fusion impaired AnPGI accumulation and both tags impaired activity, albeit to different extents. The recombinant protein showed activity against polygalacturonic acid with optimum conditions at pH 5.0 and temperature from 30 to 50°C, depending on its fusion. In vivo analysis of reducing sugar content revealed a higher release of reducing sugars in plant tissue expressing recombinant AnPGI compared to wild type N. benthamiana leaves. CONCLUSION: Our results demonstrate that subcellular compartmentalization of enzymes has an impact on both the target protein accumulation and its activity, especially in the case of proteins that undergo post-translational modifications, and should be taken into consideration when protein production strategies are designed. Using plants to produce heterologous enzymes for the degradation of a key component of the plant cell wall could reduce the cost of biomass pretreatment for the production of cellulosic biofuels.

Following vegetative to embryonic cellular changes in leaves of Arabidopsis overexpressing LEAFY COTYLEDON2.

Embryogenesis in flowering plants is controlled by a complex interplay of genetic, biochemical, and physiological regulators. LEAFY COTYLEDON2 (LEC2) is among a small number of key transcriptional regulators that are known to play important roles in controlling major events during the maturation stage of embryogenesis, notably, the synthesis and accumulation of storage reserves. LEC2 overexpression causes vegetative tissues to change their developmental fate to an embryonic state; however, little information exists about the cellular changes that take place. We show that LEC2 alters leaf morphology and anatomy and causes embryogenic structures to form subcellularly in leaves of Arabidopsis (Arabidopsis thaliana). Chloroplasts accumulate more starch, the cytoplasm fills with oil bodies, and lytic vacuoles (LVs) appear smaller in size and accumulate protein deposits. Because LEC2 is responsible for activating the synthesis of seed storage proteins (SSPs) during seed development, SSP accumulation was investigated in leaves. The major Arabidopsis SSP families were shown to accumulate within small leaf vacuoles. By exploiting the developmental and tissue-specific localization of two tonoplast intrinsic protein isoforms, the small leaf vacuoles were identified as protein storage vacuoles (PSVs). Confocal analyses of leaf vacuoles expressing fluorescently labeled tonoplast intrinsic protein isoforms reveal an altered tonoplast morphology resembling an amalgamation of a LV and PSV. Results suggest that as the LV transitions to a PSV, the tonoplast remodels before the large vacuole lumen is replaced by smaller PSVs. Finally, using vegetative and seed markers to monitor the transition, we show that LEC2 induces a reprogramming of leaf development.

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. So, it is viewed as inevitable by the One Health Initiative that humans need to reduce the use of antibiotics and turn to alternative, improved means to control disease: vaccination and prophylactics. Besides the intense research focused on novel therapeutic molecules, both these strategies rely heavily on the availability of cost-effective, efficient and scalable production platforms which will allow large-volume manufacturing for vaccines, antibodies and other biopharmaceuticals. Within this context, plant-based platforms for production of recombinant therapeutic proteins offer significant advantages over conventional expression systems, including lack of animal pathogens, low production costs, fast turnaround and response times and rapid, nearly-unlimited scalability. Also, because dried leaves and seeds can be stored at room temperature for lengthy periods without loss of recombinant proteins, plant expression systems have the potential to offer lucrative benefits from the development of edible vaccines and prophylactics, as these would not require "cold chain" storage and transportation, and could be administered in mass volumes with minimal processing. Several biotechnology companies currently have developed and adopted plant-based platforms for commercial production of recombinant protein therapeutics. In this manuscript, we outline the challenges in the process of livestock immunization as well as the current plant biotechnology developments aimed to address these challenges.

Mucosal Immunization with Spore-Based Vaccines against Mannheimia haemolytica Enhances Antigen-Specific Immunity.

BACKGROUND: Mannheimia haemolytica is a bovine respiratory pathogen commonly associated with bacterial bronchopneumonia. Current vaccine strategies have shown variable efficacy in feedlot cattle, and therefore novel vaccines are needed. Bacillus subtilis spores have been investigated as a mucosal vaccine platform, due to their ability to bind and present antigens to the mucosa and act as an adjuvant. The aim of this study was to develop two spore-based mucosal vaccines targeting M. haemolytica and evaluate their immunogenicity in mice. METHODS: Two antigen constructs composed of cholera toxin B subunit, M. haemolytica leukotoxin, and either the M. haemolytica outer membrane protein PlpE (MhCP1) or GS60 (MhCP2) were synthesized, purified and then bound to spores as vaccines. In two separate mice trials, the spore-bound vaccines (Spore-MhCP1 and Spore-MhCP2) were administered to mice through intranasal and intragastric routes, while free antigens were administered intranasally and intramuscularly. Unbound spores were also evaluated intranasally. Antigen-specific serum IgG and mucosal IgA from bronchoalveolar lavage, feces, and saliva were measured after vaccination. Mice sera from all treatment groups were assessed for their bactericidal activity against M. haemolytica. RESULTS: In both mice experiments, intramuscular immunization induced the strongest serum IgG antibody response. However, the intranasal administration of Spore-MhCP1 and Spore-MhCP2 elicited the greatest secretory IgA-specific response against leukotoxin, PlpE, and GS60 in bronchoalveolar lavage, saliva, and feces (p < 0.05). Compared to the intranasal administration of free antigen, spore-bound antigen groups showed greater bactericidal activity against M. haemolytica (p < 0.05). CONCLUSIONS: Since intranasally delivered Spore-MhCP1 and Spore-MhCP2 elicited both systemic and mucosal immune responses in mice, these vaccines may have potential to mitigate lung infection in cattle by restricting M. haemolytica colonization and proliferation in the respiratory tract. The efficacy of these mucosal spore-based vaccines merits further assessment against M. haemolytica in cattle.

Protein body formation in stable transgenic tobacco expressing elastin-like polypeptide and hydrophobin fusion proteins.

BACKGROUND: Plants are recognized as an efficient and inexpensive system to produce valuable recombinant proteins. Two different strategies have been commonly used for the expression of recombinant proteins in plants: transient expression mediated by Agrobacterium; or stable transformation of the plant genome. However, the use of plants as bioreactors still faces two main limitations: low accumulation levels of some recombinant proteins and lack of efficient purification methods. Elastin-like polypeptide (ELP), hydrophobin I (HFBI) and Zera® are three fusion partners found to increase the accumulation levels of recombinant proteins and induce the formation of protein bodies (PBs) in leaves when targeted to the endoplasmic reticulum (ER) in transient expression assays. In this study the effects of ELP and HFBI fusion tags on recombinant protein accumulation levels and PB formation was examined in stable transgenic Nicotiana tabacum. RESULTS: The accumulation of recombinant protein and PB formation was evaluated in two cultivars of Nicotiana tabacum transformed with green fluorescent protein (GFP) fused to ELP or HFBI, both targeted and retrieved to the ER. The ELP and HFBI tags increased the accumulation of the recombinant protein and induced the formation of PBs in leaves of stable transgenic plants from both cultivars. Furthermore, these tags induced the formation of PBs in a concentration-dependent manner, where a specific level of recombinant protein accumulation was required for PBs to appear. Moreover, agro-infiltration of plants accumulating low levels of recombinant protein with p19, a suppressor of post-transcriptional gene silencing (PTGS), increased accumulation levels in four independent transgenic lines, suggesting that PTGS might have caused the low accumulation levels in these plants. CONCLUSION: The use of ELP and HFBI tags as fusion partners in stable transgenic plants of tobacco is feasible and promising. In a constitutive environment, these tags increase the accumulation levels of the recombinant protein and induce the formation of PBs regardless of the cultivar used. However, a specific level of recombinant protein accumulation needs to be reached for PBs to form.

Identification of the factors that control synthesis and accumulation of a therapeutic protein, human immune-regulatory interleukin-10, in Arabidopsis thaliana.

Plants are one of the most economical platforms for large-scale production of recombinant proteins for biopharmaceutical and industrial uses. A large number of human recombinant proteins of therapeutic value have been successfully produced in plant systems. One of the main technical challenges of producing recombinant proteins in plants is to obtain sufficient level of protein. This research aims to identify the factors that control synthesis and accumulation of recombinant proteins in stable transgenic plants. A stepwise dissection of human immune-regulatory interleukin-10 (IL-10) protein production was carried out using Arabidopsis thaliana as a model system. EMS-mutagenized transgenic Arabidopsis IL-10 lines, at2762 and at3262, produced significantly higher amount of IL-10 protein than the non-mutagenized IL-10 line (WT-IL-10). The fates of trans-gene in these sets of plants were compared in detail by measuring synthesis and accumulation of IL-10 transcript, transcript stability, protein synthesis and IL-10 protein accumulation. The IL-10 transcripts were more stable in at2762 and at3262 lines than WT-IL-10, which may contribute to higher protein synthesis in these lines. To evaluate whether translational regulation of IL-10 controls its synthesis in non-mutagenized WT-IL-10 and higher IL-10 accumulating mutant lines, we measured the efficiency of the translational machinery. Our results indicate that mutant lines with higher trans-gene expression contain more robust and efficient translational machinery compared with the control line.

High-level production of human interleukin-10 fusions in tobacco cell suspension cultures.

The production of pharmaceutical proteins in plants has made much progress in recent years with the development of transient expression systems, transplastomic technology and humanizing glycosylation patterns in plants. However, the first therapeutic proteins approved for administration to humans and animals were made in plant cell suspensions for reasons of containment, rapid scale-up and lack of toxic contaminants. In this study, we have investigated the production of human interleukin-10 (IL-10) in tobacco BY-2 cell suspension and evaluated the effect of an elastin-like polypeptide tag (ELP) and a green fluorescent protein (GFP) tag on IL-10 accumulation. We report the highest accumulation levels of hIL-10 obtained with any stable plant expression system using the ELP fusion strategy. Although IL-10-ELP has cytokine activity, its activity is reduced compared to unfused IL-10, likely caused by interference of ELP with folding of IL-10. Green fluorescent protein has no effect on IL-10 accumulation, but examining the trafficking of IL-10-GFP over the cell culture cycle revealed fluorescence in the vacuole during the stationary phase of the culture growth cycle. Analysis of isolated vacuoles indicated that GFP alone is found in vacuoles, while the full-size fusion remains in the whole-cell extract. This indicates that GFP is cleaved off prior to its trafficking to the vacuole. On the other hand, IL-10-GFP-ELP remains mostly in the ER and accumulates to high levels. Protein bodies were observed at the end of the culture cycle and are thought to arise as a consequence of high levels of accumulation in the ER.

Development of a plant-based oral vaccine candidate against the bovine respiratory pathogen Mannheimia haemolytica.

Bovine respiratory disease (BRD) affects feedlot cattle across North America, resulting in economic losses due to animal treatment and reduced performance. In an effort to develop a vaccine candidate targeting a primary bacterial agent contributing to BRD, we produced a tripartite antigen consisting of segments of the virulence factor Leukotoxin A (LktA) and lipoprotein PlpE from Mannheimia haemolytica, fused to a cholera toxin mucosal adjuvant (CTB). This recombinant subunit vaccine candidate was expressed in the leaves of Nicotiana benthamiana plants, with accumulation tested in five subcellular compartments. The recombinant protein was found to accumulate highest in the endoplasmic reticulum, but targeting to the chloroplast was employed for scaling up production due the absence of post-translational modification while still producing feasible levels. Leaves were freeze dried, then orally administered to mice to determine its immunogenicity. Sera from mice immunized with leaf tissue expressing the recombinant antigen contained IgG antibodies, specifically recognizing both LktA and PlpE. These mice also had a mucosal immune response to the CTB+LktA+PlpE protein as measured by the presence of LktA- and PlpE-specific IgA antibodies in lung and fecal material. Moreover, the antigen remained stable at room temperature with limited deterioration for up to one year when stored as lyophilized plant material. This study demonstrated that a recombinant antigen expressed in plant tissue elicited both humoral and mucosal immune responses when fed to mice, and warrants evaluation in cattle.

Plant production of a virus-like particle-based vaccine candidate against porcine reproductive and respiratory syndrome.

Porcine reproductive and respiratory syndrome (PRRS) is a disease leading to spontaneous abortions and stillbirths in sows and lowered life quality and expectancy in growing pigs. PRRS is prevalent worldwide and has significant economic impacts to swine industries around the globe. Co-expression of the two most abundant proteins in the viral envelope, the matrix protein (M) and glycosylated protein 5 (GP5), can produce a neutralizing immune response for the virus providing a potentially effective subunit vaccine against the disease, but these proteins are difficult to express. The goal of this research was to display antigenic portions of the M and GP5 proteins on the surface of tobacco mosaic virus-like particles. A modified tobacco mosaic virus coat protein (TMVc) was transiently expressed in Nicotiana benthamiana leaves and targeted to three subcellular compartments along the secretory pathway to introduce glycosylation patterns important for M-GP5 epitope immunogenicity. We found that accumulation levels in the apoplast were similar to the ER and the vacuole. Because glycans present on plant apoplastic proteins are closest to those present on PRRSV proteins, a TMVc-M-GP5 fusion construct was targeted to the apoplast and accumulated at over 0.5 mg/g of plant fresh weight. TMVc virus-like particles self-assembled in plant cells and surface-displayed the M-GP5 epitope, as visualized by transmission electron microscopy and immunogold localization. These promising findings lay the foundation for immunogenicity and protective-immunity studies in animals to examine the efficacy of this vaccine candidate as a measure to control PRRS.

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. We also describe a protocol for detection of full-length dsRNA molecules in plastids using an RT-PCR-based method.

Protein body-inducing fusions for high-level production and purification of recombinant proteins in plants.

For the past two decades, therapeutic and industrially important proteins have been expressed in plants with varying levels of success. The two major challenges hindering the economical production of plant-made recombinant proteins include inadequate accumulation levels and the lack of efficient purification methods. To address these limitations, several fusion protein strategies have been recently developed to significantly enhance the production yield of plant-made recombinant proteins, while simultaneously assisting in their subsequent purification. Elastin-like polypeptides are thermally responsive biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the purification of recombinant proteins. Hydrophobins are small fungal proteins capable of altering the hydrophobicity of their respective fusion partner, thus enabling efficient purification by surfactant-based aqueous two-phase systems. Zera, a domain of the maize seed storage protein γ-zein, can induce the formation of protein storage bodies, thus facilitating the recovery of fused proteins using density-based separation methods. These three novel protein fusion systems have also been shown to enhance the accumulation of a range of different recombinant proteins, while concurrently inducing the formation of protein bodies. The packing of these fusion proteins into protein bodies may exclude the recombinant protein from normal physiological turnover. Furthermore, these systems allow for quick, simple and inexpensive nonchromatographic purification of the recombinant protein, which can be scaled up to industrial levels of protein production. This review will focus on the similarities and differences of these artificial storage organelles, their biogenesis and their implication for the production of recombinant proteins in plants and their subsequent purification.

Recombinant protein production in a variety of Nicotiana hosts: a comparative analysis.

Although many different crop species have been used to produce a wide range of vaccines, antibodies, biopharmaceuticals and industrial enzymes, tobacco has the most established history for the production of recombinant proteins. To further improve the heterologous protein yield of tobacco platforms, transient and stable expression of four recombinant proteins (i.e. human erythropoietin and interleukin-10, an antibody against Pseudomonas aeruginosa, and a hyperthermostable α-amylase) was evaluated in numerous species and cultivars of Nicotiana. Whereas the transient level of recombinant protein accumulation varied significantly amongst the different Nicotiana plant hosts, the variety of Nicotiana had little practical impact on the recombinant protein concentration in stable transgenic plants. In addition, this study examined the growth rate, amount of leaf biomass, total soluble protein levels and the alkaloid content of the various Nicotiana varieties to establish the best plant platform for commercial production of recombinant proteins. Of the 52 Nicotiana varieties evaluated, Nicotiana tabacum (cv. I 64) produced the highest transient concentrations of recombinant proteins, in addition to producing a large amount of biomass and a relatively low quantity of alkaloids, probably making it the most effective plant host for recombinant protein production.

Hydrophobin fusions for high-level transient protein expression and purification in Nicotiana benthamiana.

Insufficient accumulation levels of recombinant proteins in plants and the lack of efficient purification methods for recovering these valuable proteins have hindered the development of plant biotechnology applications. Hydrophobins are small and surface-active proteins derived from filamentous fungi that can be easily purified by a surfactant-based aqueous two-phase system. In this study, the hydrophobin HFBI sequence from Trichoderma reesei was fused to green fluorescent protein (GFP) and transiently expressed in Nicotiana benthamiana plants by Agrobacterium tumefaciens infiltration. The HFBI fusion significantly enhanced the accumulation of GFP, with the concentration of the fusion protein reaching 51% of total soluble protein, while also delaying necrosis of the infiltrated leaves. Furthermore, the endoplasmic reticulum-targeted GFP-HFBI fusion induced the formation of large novel protein bodies. A simple and scalable surfactant-based aqueous two-phase system was optimized to recover the HFBI fusion proteins from leaf extracts. The single-step phase separation was able to selectively recover up to 91% of the GFP-HFBI up to concentrations of 10 mg mL(-1). HFBI fusions increased the expression levels of plant-made recombinant proteins while also providing a simple means for their subsequent purification. This hydrophobin fusion technology, when combined with the speed and posttranslational modification capabilities of plants, enhances the value of transient plant-based expression systems.