Proteins In Plant Tissues Research Articles

How does ionizing radiation affect amyloidogenesis in plants?

Purpose Ionizing radiation is a harsh environmental factor that could induce plant senescence. We hypothesized that radiation-related senescence remodels proteome, particularly by triggering the accumulation of prion-like proteins in plant tissues. The object of this study, pea (Pisum sativum L.), is an agriculturally important legume. Research on the functional importance of amyloidogenic proteins was never performed on this species. Materials and methods Pea seeds were irradiated in the dose range 5–50 Gy of X-rays. Afterward, Fourier-transform infrared spectroscopy (FTIR) was used to investigate changes in the secondary structure of proteins in germinated 3-day-old seedlings. Specifically, we evaluated the ratio between the amide I and II peaks. Next, we performed protein staining with Congo red to compare the presence of amyloids in the samples. In parallel, we profiled the detergent-resistant proteome fraction by ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS). Differentially accumulated proteins were functionally analyzed in MapMan software, and the PLAAC tool was used to predict putative prion-like proteins. Results We showed a reduced germination rate but higher plant height and faster appearance of reproductive organs in the irradiated at dose of 50 Gy group compared with the control; furthermore, we demonstrated more β-sheets and amyloid aggregates in the roots of stressed plants. We detected 531 proteins in detergent-resistant fraction extracted from roots, and 45 were annotated as putative prion-like proteins. Notably, 29 proteins were significantly differentially abundant between the irradiated and the control groups. These proteins belong to several functional categories: amino acid metabolism, carbohydrate metabolism, cytoskeleton organization, regulatory processes, protein biosynthesis, and RNA processing. Thus, the discovery proteomics provided deep data on novel aspects of plant stress biology. Conclusion Our data hinted that protein accumulation stimulated seedlings’ growth as well as accelerated ontogenesis and, eventually, senescence, primarily through translation and RNA processing. The increased abundance of primary metabolism-related proteins indicates more intensive metabolic processes triggered in germinating pea seeds upon X-ray exposure. The functional role of detected putative amyloidogenic proteins should be validated in overexpression or knockout follow-up studies.

Obtaining of transgenic barrelclover plants (Medicago truncatula) producing chicken interferon gamma for veterinary use

At the Laboratory of Plant Genetic and Cellular Engineering, Department of Genetics and Biotechnology, St. Petersburg State University, five transgenic Medicago truncatula plants were obtained through Agrobacterium-mediated transformation, carrying one of the variants of the heterologous chicken interferon-gamma gene under the control of the constitutive 35S CaMV promoter. Among these, one plant harbored an unmodified gene insertion, while four had a modified gene with a deletion at the protease recognition site, providing resistance to proteolytic degradation.
 We demonstrate the application of the SWPOP-PCR “genome walking” method to determine the integration sites of T-DNA into the plant genome, identify the number of insertion copies and their orientation. Analysis of the obtained sequences revealed that only one plant exhibited a single T-DNA insertion, which represents the most optimal structure for stable expression.
 Upon self-pollination of T0 plants, 39 offspring were obtained and subjected to testing for the presence and expression of the transgene. Among them, six homozygous plants were identified using molecular methods. Quantitative assessment of transgene expression levels showed significant differences among representatives of different lines and among the offspring derived from a single transformed plant. Among the T1 and T2 progeny, the presence of the heterologous interferon protein in plant tissues was confirmed through Western blot analysis.
 The engineered barrelclover plants hold potential as bioreactors for the production of chicken interferon-gamma for veterinary applications. The use of an edible plant allows eliminating protein extraction and purification procedures, thereby resulting in a noteworthy reduction in production expenses of up to 80%.

Biochemical and proteomic insights revealed selenium priming induced phosphorus stress tolerance in common bean (Phaseolus vulgaris L.).

Mineral stress is one of the dominating abiotic stresses, which leads to decrease in crop production. Selenium (Se) seed priming is a recent approach to mitigate the plant's mineral deficiency stress. Although not an essential element, Se has beneficial effects on the plants in terms of growth, quality, yield and plant defense system thus, enhancing plant tolerance to mineral deficiency. The present research was accomplished to find out the effect of Se primingon common beanplant (SFB-1 variety) under phosphorus (P) stress. The seeds were growninvitroon four different MGRL media which are normal MGRL media as control with non-Se primed seeds (Se-P+), non -Se primed seeds grown on P deficient MGRL media (Se-P-), Se primed seeds grown on normal MGRL media (Se+P+) and Se primed seeds grown on P deficient MGRL media (Se+P-). The various morphological and biochemical parameters such as proline content, total sugar content, polyphenols and expression of proteins were analyzed under P stress.The results showed that Se priming has significantly (p ≤ 0.05) affected the morphological as well as biochemical parameters under normal and P stress conditions. The morphological parameters-length, weight, number of nodes and leaves of Se+P+, Se+P- root and shoot tissue showed significant increase as compared to Se-P+,Se-P-.Similarly variousbiochemical parameters such as total chlorophyll content, proline, total sugar content and polyphenols of Se+P+, Se+P-increased significantly as compared to Se-P+,Se-P-. The differential protein expression in both Se+P+, Se+P- andSe-P+, Se-P-plants were determinedusingMALDI-MS/MS. Thedifferentially expressed proteinsinSe+P+, Se+P-plants were identified as caffeic acid-3-O-methyltransferase (COMT) and SecA protein (a subunit of Protein Translocan transporter), and are found responsible for lignin synthesis in root cell walls and ATP dependent movement of thylakoid proteins across the membranes in shoot respectively. The differential expression of proteins in plant tissues, validated morphological and biochemical responses such as maintaining membrane integrity, enhanced modifications in cellular metabolism, improved polyphenol activities and expression of defensive proteins against mineral deficiency. The study provided an understanding of Se application as a potential approach increasing tolerance and yield in crop plants against mineral deficiency.

Interaction between sulfur and selenium in agronomic biofortification of cowpea plants under field conditions

AimsSelenium (Se) as selenate shares similarities with sulfate in transport and assimilation by plants. Uptake and assimilation of Se might be affected by S and vice-versa, which could affect Se and S concentration in plant tissues, and metabolic pathways such as biosynthesis of sugars, amino acids, and storage proteins. This study aimed to evaluate Se and S combination on cowpea plants under field conditions.MethodsThe experimental design was a 4 × 4 interaction between four rates of Se (0, 10, 25, and 50 g ha− 1) and four rates of S (0, 15, 30, and 60 kg ha− 1) in two consecutive years of cowpea cultivation. Concentrations of Se, S, total sugars, sucrose, total free amino acids, and storage proteins in plant tissue were measured.ResultsThe Se x S interaction did not affect cowpea yield or growth. Antagonistic effects of S on Se concentrations in leaves and seeds were observed mainly for the second crop season. Selenium did not decrease S concentrations in leaves and seeds of cowpea plants. The combination of 25 g Se ha− 1 and 30 kg S ha− 1 provided the greater concentrations of total sugars. Interaction between Se and S was associated with greater sucrose, amino acids, and storage proteins concentrations in cowpea seeds.ConclusionsThe Se and S interaction did not impair plant growth but application of S decreased Se content in cowpea. Further studies are needed to better understand the physiological roles of Se and S combination in producing primary metabolic compounds.

光 - 電子相関顕微鏡法とアレイトモグラフィー法を組み合わせて蛍光を放つオルガネラを電顕で捉える

Correlative light and electron microscopy (CLEM) is an analytical technique used to evaluate the correlation between fluorescence images of identical samples and regions of fluorescent-labeled organelles captured using light microscopy and electron microscopy. By utilizing various methods during sample preparation, the fluorescence images of fluorescent-labeled organelles are juxtaposed with ultra-structures of the same region obtained using scanning electron microscopy (SEM) or transmission electron microscopy. Unlike animal tissues and adherent cells, visualizing the organelles detected in plant samples via CLEM analysis is difficult because of the non-adherence of plant tissue and cultured cells to glass plates and the size of the cells. Therefore, we combine the serial section SEM method (Array tomography) based on ultra-thin sections and CLEM to identify fluorescent-labeled organelles in plant cells and reconstruct them in three dimensions (3D). In particular, as osmium (Os) epoxy-tolerant fluorescent proteins have recently been reported to be resistant to Os fixation and epoxy resin embedding in animal adherent cells, we applied these fluorescent tags to plant samples. In this paper, we introduce the sample preparation method for the CLEM analysis of organelles labeled with Os epoxy-tolerant fluorescent proteins in plant tissues, alongside the method for obtaining CLEM images and the combined 3D-CLEM analysis method comprising CLEM and array tomography.

Electrophoretic Detection and Confocal Microscopic Imaging of Tyrosine Nitrated Proteins in Plant Tissue.

Tyrosine nitrated proteins can be detected in plant cells electrophoretically and their distribution can be monitored by confocal laser scanning microscopy (CLSM) imaging. One-dimensional polyacrylamide gel electrophoresis (1D PAGE) followed by Western blotting using polyclonal antibody against 3-nitrotyrosine residues enables detection of tyrosine nitrated proteins in plant cells. Here we describe detection of tyrosine nitrated proteins in the homogenates derived from sunflower (Helianthus annuus L.) seedling cotyledons. Total soluble proteins obtained from tissue homogenates are resolved using vertical gel electrophoresis followed by their electrophoretic transfer on to a microporous membrane support for immunodetection. Spatial distribution of tyrosine nitrated proteins can be visualized using an antibody against 3-nitrotyrosine residues. Immunofluorescent localization is performed by cutting 7μm thick wax sections of tissue followed by incubation in primary anti-nitrotyrosine antibody (dilution 1:200) and secondary Cy-3 labeled anti-rabbit IgG antibody (dilution 1:1500). Confocal laser scanning microscopy analysis is undertaken using argon lasers (ex: 530-550 nm and em: 570nm) at pinhole 1. Modulation in the abundance and spatial localization of tyrosine nitrated proteins in plant tissues can be monitored using these techniques.

Characterization of UDP-glucosyltransferase from Indigofera tinctoria

Indican is a secondary metabolite in Indigofera tinctoria; its synthesis from indoxyl and UDP-glucose is catalyzed by a UDP-glucosyltransferase (UGT). In this study, we partially purified UGT extracted from I. tinctoria leaves and analyzed the protein by peptide mass fingerprinting. We identified two fragments that were homologous to UGT after comparison with the transcriptomic data of I. tinctoria leaves. The fragments were named itUgt1 and itUgt2 and were amplified using rapid amplification of cDNA ends polymerase chain reaction to obtain full-length cDNAs. The resultant nucleotide sequences of itUgt1 and itUgt2 encoded peptides of 477 and 475 amino acids, respectively. The primary structure of itUGT1 was 89% identical to that of itUGT2 and contained an important plant secondary product glycosyltransferase (PSPG) box sequence and a UGT motif.The recombinant proteins expressed in Escherichia coli were found to possess high indican synthesis activity. Although the properties of the two proteins itUGT1 and itUGT2 were very similar, itUGT2 was more stable at high temperatures than itUGT1. Expression levels of itUGT mRNA and protein in plant tissues were examined by UGT assay, immunoblotting, and semi-quantitative reverse transcription polymerase chain reaction. So far, we presume that itUGT1, but not itUGT2, primarily catalyzes indican synthesis in I. tinctoria leaves.

Dimethyl-Labeling-Based Quantification of the Lysine Acetylome and Proteome of Plants.

Photorespiratory enzymes in different cellular compartments have been reported to be posttranslational modified by phosphorylation, disulfide formation, S-nitrosylation, glutathionylation, and lysine acetylation. However, not much is known yet about the function of these modifications to regulate the activities, localizations, or interactions of the proteins in this metabolic pathway. Hence, it will be of great importance to study these modifications and their temporal and conditional occurrence in more detail. Here, we focus on the analysis of lysine acetylation as a relatively newly discovered modification on plant metabolic enzymes. The acetylation of lysine residues within proteins is a highly conserved and reversible posttranslational modification which occurs in all living organisms. First discovered on histones and implied in the regulation of gene expression, lysine acetylation also occurs on a diverse set of cellular proteins in different subcellular compartments and is particularly abundant on metabolic enzymes. Upon lysine acetylation, the function of proteins can be modulated due to the loss of the positive charge of the lysine residue. Lysine acetylation was also discovered on proteins involved in photosynthesis and novel tools are needed to study the regulation of this modification in dependence on the environmental conditions, tissues, or plant genotype. This chapter describes a method for the identification and relative quantification of lysine-acetylated proteins in plant tissues using a dimethyl labeling technique combined with an anti-acetyl lysine antibody enrichment strategy. Here, we describe the protein purification, labeling of trypsinated peptides, as well as immuno-enrichment of lysine-acetylated peptides followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) data acquisition and analysis.

Overexpression of TAT-PTD-diapause hormone fusion protein in tobacco and its effect on the larval development of Helicoverpa armigera (Lepidoptera: Noctuidae).

The diapause hormone (DH) has been shown either to induce or to terminate diapause, depending on the insect species. In a previous study we demonstrated that the DH from Clostera anastomosis (caDH) has biological activity in Helicoverpa armigera, which prompted us to examine the potential growth-inhibiting or antiherbivory effects of the TAT-caDH fusion protein when expressed in transgenic plants. In this study, we produced transgenic tobacco plants expressing either the TAT-caDH protein or a TAT-caDH-eGFP fusion version that allowed tracking of the fluorescent protein in plant tissues. Our results indicate that H. armigera larvae feeding on transgenic tobacco expressing TAT-caDH exhibited a significantly reduced survival rate and weight gain. However, larvae feeding on transgenic tobacco expressing TAT-caDH-eGFP were unaffected. While fusion of the eGFP gene influenced the bioactivity of caDH in larvae, TAT-caDH-eGFP can still penetrate the insect midgut cell membrane. TAT-caDH increases DH stability in oral delivery. Our results may help in targeting DH-dependent physiological processes in insects for improving herbivore tolerance in economically important crops. © 2016 Society of Chemical Industry.

The spatial distribution of phosphate in the root system modulates N metabolism and growth in Eucalyptus grandis young plants

High P homogeneously applied in roots reduced growth and nitrate uptake. High P in a patch reduced N in tissues but did not reduce growth, because root hydraulic conductivity increased. Little is known about consequences of nutrient imbalances, i.e., when the increase of one nutrient’s availability, in non-toxic concentration, reduces plant growth, because other nutrient availability is low. In a soil with low N (nitrogen) availability, high homogeneous P (phosphorus) availability reduced nitrate uptake and reduced growth of Eucalyptus grandis plants. However, if the same availability of P was applied only in a part of the root system (localized), no depressive effect was observed. In this work, it was analyzed which step in the early assimilation of N was affected by high homogeneous P and how localized P counteracted this depressive effect. Inorganic forms of N and protein in plant tissues as well as some plant hydraulic traits were analyzed in an experiment with E. grandis plants growing in perlite in a split-root system fed with low N. Control plants received low P. High P was applied in two spatial distributions: localized in one part of the root system (LP) or distributed homogeneously in both parts (HP). HP reduced growth, while LP had no depressive effect in growth. Both high P spatial distributions reduced concentration of nitrate in roots. Since concentration of nitrate in the xylem was similar in all treatments and nitrate in leaves was lower in high P than in control treatment, the reduction in root N was probably due to lower uptake. Nevertheless, plants growing with LP had no depressive effect in growth, because the decrease in N assimilation was counteracted by an increase of root hydraulic conductivity.

Rapid labeling of intracellular His-tagged proteins in living cells

Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni(2+)-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni(2+)-NTA-based probes. Unfortunately, previous Ni-NTA-based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni(2+) ions. The probe, driven by Ni(2+)-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to be mainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells.

Visualization of hormone binding proteins in vivo based on Mn-doped CdTe QDs

Daminozide (B9) is a growth inhibitor with important regulatory roles in plant growth and development. Locating and quantifying B9-binding proteins in plant tissues will assist in investigating the mechanism behind the signal transduction of B9. In this study, red fluorescent Mn-doped CdTe quantum dots (CdTeMn QDs) were synthesized by a high-temperature hydrothermal process. Since CdTeMn QDs possess a maximum fluorescence emission peak at 610nm, their fluorescence properties are more stable than those of CdTe QDs. A B9-CdTeMn probe was synthesized by coupling B9 with CdTeMn QDs. The fluorescence intensity of the probe is double that of CdTeMn QDs; its fluorescence stability is also superior under different ambient conditions. The probe retains the biological activity of B9 and is unaffected by interference from the green fluorescent protein present in plants. Therefore, we used this probe to label B9-binding proteins selectively in root tissue sections of mung bean seedlings. These proteins were observed predominantly on the surfaces of the cell membranes of the cortex and epidermal parenchyma.

Attachment of Agrobacterium tumefaciens to leaf tissue in response to infiltration conditions.

Transient expression of recombinant proteins in plant tissues following Agrobacterium-mediated gene transfer is a promising technique for rapid protein production. However, transformation rates and transient expression levels can be sub-optimal depending on process conditions. Attachment of Agrobacterium tumefaciens to plant cells is an early, critical step in the gene transfer pathway. Bacterial attachment levels and patterns may influence transformation and, by extension, transient expression. In this study, attachment of A. tumefaciens to lettuce leaf tissue was investigated in response to varying infiltration conditions, including bacterial density, surfactant concentration, and applied vacuum level. Bacterial density was found to most influence attachment levels for the levels tested (10(8) , 10(9) , and 10(10) CFU/mL), with the relationship between bacterial density and attachment levels following a saturation trend. Surfactant levels tested (Break-Thru S240: 1, 10, 100, and 1,000 µL/L) also had a significant positive effect on bacterial attachment while vacuum level (5, 25, and 45 kPa) did not significantly affect attachment in areas exposed to bacteria. In planta transgene transient expression levels were measured following infiltration with 10(8) , 10(9) , and 10(10) CFU/mL bacterial suspension. Notably, the highest attachment level tested led to a decrease in transient expression, suggesting a potential link between bacterial attachment levels and downstream phenomena that may induce gene silencing. These results illustrate that attachment can be controlled by adjusting infiltration conditions and that attachment levels can impact transgene transient expression in leaf tissue.

Applications of Plant-derived Vaccines for Developing Countries

Plants offer tremendous advantages as cost-effective, safe and efficacious platforms for the large-scale production of vaccines and other therapeutic proteins. Plant-derived vaccines provide a way by which to enhance vaccine coverage for children in developing countries, and have the potential via oral administration to elicit a mucosal immune response. Plants have the added advantage of simultaneously acting as an antigen delivery vehicle to the mucosal immune system while preventing the antigen from degradation as it passes through the gastrointestinal tract. Transgenic plants, transplastomic plants and plant virus expression vectors have been designed to express vaccine epitopes as well as full therapeutic proteins in plant tissue. This review describes the use of different strategies to produce vaccines in plants against three deadly infectious diseases of the developing world today; human papillomavirus, human immunodeficiency virus and Ebola virus.

Recent Advances in Virus Expression Vector Strategies for Vaccine Production in Plants

Plants offer tremendous advantages as cost-effective and safe platforms for the large-scale production of vaccines and other therapeutic proteins. Plant-derived vaccines represent a means by which to enhance vaccine coverage for children in developing countries, and can be administered orally to elicit a mucosal immune response. Plantderived vaccines possess the dual advantage of preventing the antigen from degradation as it passes through the gastrointestinal tract, while at the same time being capable of delivering an antigen to the mucosal immune system. Plant virus vectors have been designed to express vaccine epitopes as well as full therapeutic proteins in plant tissue. This review describes recent advances with respect to plant virus expression vectors used as production platforms for biopharmaceutical proteins.

Variability of Endotoxin Expression in Bt Transgenic Cotton

AbstractTransgenic cotton expressing Bt (Bacillus thuringiensis) toxins is currently cultivated on a large commercial scale in many countries, but observations have shown that it behaves variably in toxin efficacy against target insects under field conditions. Understanding of the temporal and spatial variation in efficacy and the resulting mechanisms is essential for cotton protection and production. In this review, we summarize current knowledge on variability in Bt cotton efficacy, in particular on the induced variability by environmental stresses. We also discuss the resulting mechanisms and the countermeasures for the inconsistence in efficacy in Bt cotton. It is indicated that insecticidal protein content in Bt cotton is variable with plant age, plant structure or under certain environmental stresses. Variability in Bt cotton efficacy against target insect pests is mainly attributed to the changes in Bt protein content, but physiological changes associated with the production of secondary compounds in plant tissues may also play an important role. Reduction of Bt protein content in late‐season cotton could be due to the overexpression of Bt gene at earlier stages, which leads to gene regulation at post‐transcription levels and consequently results in gene silencing at a later stage. Methylation of the promotor may be also involved in the declined expression of endotoxin proteins. As a part of total protein, the insecticidal protein in plant tissues changes its level through inhibited synthesis, degradation or translocation to developing plant parts, particularly under environmental stresses, thus being closely correlated to N metabolism. It can be concluded that developing new cotton varieties with more powerful resistance, applying certain plant growth regulators, enhancing intra‐plant defensive capability, and maintenance of general health of the transgenic crop are important in realizing the full transgenic potential in Bt cotton.

A chemically inducible cucumber mosaic virus amplicon system for expression of heterologous proteins in plant tissues

A novel cucumber mosaic virus inducible viral amplicon (CMViva) expression system has been developed that allows for tightly regulated chemically inducible expression of heterologous genes in plant hosts. Transient production of recombinant alpha(1)-antitrypsin (rAAT), a human blood protein, was demonstrated in Nicotiana benthamiana leaves. The highest production levels were obtained by co-infiltrating leaves with Agrobacterium tumefaciens cells containing CMViva carrying the AAT gene and A. tumefaciens cells carrying a binary vector constitutively expressing the gene silencing suppressor p19. Accumulation of up to thirty-fold more rAAT was observed in leaves (24 mg per 100 g leaf tissue) when compared with the expression levels observed using the cauliflower mosaic virus (CaMV) 35S promoter. Significantly, 70% of the rAAT produced using the CMViva expression system was found to be biologically active, a 170-fold increase in functional protein compared with the CaMV 35S expression system.

Delivery of plant-derived vaccines

Many protein subunit vaccine candidates have been expressed in transgenic plants, and in a few cases the recombinant material has entered early phase clinical or target animal trials. The expressed protein can be purified prior to formulation for any preferred delivery approach. However, there are major cost advantages associated with avoiding protein purification and pursuing the oral delivery of a processed plant product containing the recombinant protein. Grains and dry products that are processed from fresh plant tissues can stably store expressed proteins for extended periods of time at room temperature, making refridgeration unnecessary during storage and distribution. Encapsulation of recombinant proteins in plant tissues guards against their rapid degradation in the gut, therefore facilitating the uptake and induction of appropriate immune responses. Early trial data with plant-based vaccine candidates has shown promising safety and efficacy.

Expression and Purification of Heterologous Proteins in Plant Tissue Using a Geminivirus Vector System

In the past, plant molecular biologists have relied on Escherichia coli, baculovirus and other expression systems to produce plant proteins to quantities sufficient for biochemical analysis. However, such expression systems often result in the production of proteins which possess improper posttranslational modifications. Here, we present a plant virus-based expression system superior to those currently available. We demonstrate that bean yellow dwarf geminivirus (BeYDV) replicates and expresses foreign proteins at high levels in tobacco, Arabidopsis, and other dicotyledonous plants, making it more universal than plant RNA viruses with restricted host ranges which are currently used as expression systems. The DNA-based nature of the BeYDV genome renders it stable for the incorporation of large plant open reading frames, and gives it an advantage over other plant virus-based expression systems which possess insert size restrictions. Using this expression system, the rapid accumulation of a novel Arabidopsis-derived mitogen-activated protein kinase to levels sufficient for standard biochemical analysis is demonstrated.

A plant signal peptide-hepatitis B surface antigen fusion protein with enhanced stability and immunogenicity expressed in plant cells.

The use of transgenic plants to express orally immunogenic protein antigens is an emerging strategy for vaccine biomanufacturing and delivery. This concept has particular suitability for developing countries. One factor that has limited the development of this technology is the relatively modest levels of accumulation of some antigenic proteins in plant tissues. We used fusion protein design to improve expression of the hepatitis B surface antigen (HBsAg) by attempting to mimic the process of HBsAg targeting to the endoplasmic reticulum of human liver cells during hepatitis B virus infection. We created a gene encoding a recombinant HBsAg modified to contain a plant signal peptide fused to its amino terminus. The signal peptide from soybean vegetative storage protein vspA (VSP alpha S) directed endoplasmic reticulum targeting of HBsAg in plant cells, but was not cleaved and resulted in enhanced VSP alpha S-HBsAg fusion accumulation. This product was more stable and presented the protective "a" antigenic determinant to significantly higher levels than unmodified native HBsAg expressed in plant cells. It also showed a greater extent of intermolecular disulfide bond formation and formation of virus-like particles. Moreover, VSP alpha S-HBsAg stimulated higher levels of serum IgG than native HBsAg when injected into mice. We conclude that HBsAg tolerates a polypeptide fusion at the amino terminus and that VSP alpha S-HBsAg is an improved antigen for plant-based expression of a subunit vaccine for hepatitis B virus.