High expression level of a foot and mouth disease virus epitope in tobacco transplastomic plants

Chloroplast transformation has an extraordinary potential for antigen production in plants because of the capacity to accumulate high levels of recombinant proteins and increased biosafety due to maternal plastid inheritance in most crops. In this article, we evaluate tobacco chloroplasts transformation for the production of a highly immunogenic epitope containing amino acid residues 135–160 of the structural protein VP1 of the foot and mouth disease virus (FMDV). To increase the accumulation levels, the peptide was expressed as a fusion protein with the β-glucuronidase reporter gene (uidA). The recombinant protein represented the 51% of the total soluble proteins in mature leaves, a level higher than those of the Rubisco large subunit, the most abundant protein in the leaf of a wild-type plant. Despite this high accumulation of heterologous protein, the transplastomic plants and wild-type tobacco were phenotypically indistinguishable. The FMDV epitope expressed in transplastomic plants was immunogenic in mice. These results show that transplastomic tobacco express efficiently the recombinant protein, and we conclude that this technology allows the production of large quantities of immunogenic proteins.     

Expression of a multi-epitope DPT fusion protein in transplastomic tobacco plants retains both antigenicity and immunogenicity of all three components of the functional oligomer

Expression of genes in plant chloroplasts provides an opportunity for enhanced production of target proteins. We report the introduction and expression of a fusion DPT protein containing immunoprotective exotoxin epitopes of Corynebacterium diphtheriae, Bordetella pertussis, and Clostridium tetani in tobacco chloroplasts. Using biolistic-mediated transformation, a plant-optimized synthetic DPT gene was successfully transferred to tobacco plastomes. Putative transplastomic T0 plants were identified by PCR, and Southern blot analysis confirmed homoplasmy in T1 progeny. ELISA assays demonstrated that the DPT protein retained antigenicity of the three components of the fusion protein. The highest level of expression in these transplastomic plants reached 0.8% of total soluble protein. To assess whether the functional recombinant protein expressed in tobacco plants would induce specific antibodies in test animals, a mice feeding experiment was conducted. For mice orally immunized with freeze-dried transplastomic leaves, production of IgG and IgA antibodies specific to each toxin were detected in serum and mucosal tissues. R. E. Soria-Guerra and A. G. Alpuche-Solís contributed equally to this work.     

Chloroplast tubules visualized in transplastomic plants expressing green fluorescent protein

A fusion between the plastid psbA promoter and the green fluorescent protein gene (gfp) was introduced into the tobacco chloroplast genome by stable plastid transformation. GFP was synthesized actively and exclusively in the chloroplasts. Tubular projections filled with GFP but containing no chlorophyll were visualized for the first time in chloroplasts of these transplastomic plants. Occasionally, the tubules connect chloroplasts with each other, suggesting the possibility of the exchange of endogenous proteins. However, the fusion of protoplasts between the transplastomic and wild-type plants showed that such chloroplast connections might be rare in mesophyll protoplasts.     

Accumulation of recombinant SARS-CoV spike protein in plant cytosol and chloroplasts indicate potential for development of plant-derived oral vaccines

Plants are promising candidates as bioreactors for the production of oral recombinant proteins in the biopharmaceutical industry. As an initial step toward provision of an oral vaccine against the severe acute respiratory syndrome coronavirus (SARS-CoV), we have expressed a partial spike (S) protein of SARS-CoV in the cytosol of nuclear-transformed plants and in the chloroplasts of plastid-transformed plants. In the construction of both nuclear and plastid transformation vectors, a 2-kilobase nucleotide sequence encoding amino acids 1-658 of the SARS-CoV spike protein (S1) was modified with nucleotide changes, but not amino acid changes, to optimize codon usage for expression in plants. To investigate the subcellular localization of S1 during transient expression in tobacco leaves, a translational fusion consisting of S1 and the green fluorescent protein (GFP) was generated. Following agroinfiltration of tobacco leaves, analysis by laser confocal scanning microscopy revealed that the S1:GFP fusion protein was localized to the cytosol. In stable transgenic tobacco plants and lettuce plants generated by Agrobacterium-mediated transformation, tobacco and lettuce leaves were observed to express the S1 at high levels from the Cauliflower Mosaic Virus 35S promoter with Northern blot analysis. When the S1 was expressed in transplastomic tobacco, S1 messenger RNA and its corresponding protein were detected on Northern and Western blot analyses, respectively. Our results demonstrate the feasibility of producing S1 in nuclear- and chloroplast-transformed plants, indicating its potential in subsequent development of a plant-derived and safe oral recombinant subunit vaccine against the SARS-CoV in edible plants.     

Improved heterologous protein expression in the chloroplast of Chlamydomonas reinhardtii through promoter and 5' untranslated region optimization

Microalgae have the potential to be a valuable biotechnological platform for the production of recombinant proteins. However, because of the complex regulatory network that tightly controls chloroplast gene expression, heterologous protein accumulation in a wild-type, photosynthetic-competent algal chloroplast remains low. High levels of heterologous protein accumulation have been achieved using the psbA promoter/5' untranslated region (UTR), but only in a psbA-deficient genetic background, because of psbA/D1-dependent auto-attenuation. Here, we examine the effect of fusing the strong 16S rRNA promoter to the 5' UTR of the psbA and atpA genes on transgene expression in the chloroplast of Chlamydomonas reinhardtii. We show that fusion of the 16S promoter had little impact on protein accumulation from the psbA 5' UTR in a psbA-deficient genetic background. Furthermore, the 16S/psbA promoter/UTR fusion was silenced in the presence of wild-type levels of D1 protein, confirming that the psbA 5' UTR is the primary target for D1-dependent auto-repression. However, fusion of the 16S promoter to the atpA 5' UTR significantly boosts mRNA levels and supports high levels of heterologous protein accumulation in photosynthetic-competent cells. The 16S/atpA promoter/UTR drove LUXCT protein accumulation to levels close to that of psbA in a psbA- background, and drove expression of a human therapeutic protein to levels only twofold lower than the psbA 5' UTR. The 16S/atpA promoter/UTR combination should have utility for heterologous protein production when expression from a photosynthetic-competent microalgal strain is required.     

Production of biologically active human thioredoxin 1 protein in lettuce chloroplasts

The production of human therapeutic proteins in plants provides opportunities for low-cost production, and minimizes the risk of contamination from potential human pathogens. Chloroplast genetic engineering is a particularly promising strategy, because plant chloroplasts can produce large amounts of foreign target proteins. Oxidative stress is a key factor in various human diseases. Human thioredoxin 1 (hTrx1) is a stress-induced protein that functions as an antioxidant against oxidative stress, and overexpression of hTrx1 has been shown to suppress various diseases in mice. Therefore, hTrx1 is a prospective candidate as a new human therapeutic protein. We created transplastomic lettuce expressing hTrx1 under the control of the psbA promoter. Transplastomic plants grew normally and were fertile. The hTrx1 protein accumulated to approximately 1% of total soluble protein in mature leaves. The hTrx1 protein purified from lettuce leaves was functionally active, and reduced insulin disulfides. The purified protein protected mouse insulinoma line 6 cells from damage by hydrogen peroxide, as reported previously for a recombinant hTrx1 expressed in Escherichia coli. This is the first report of expression of the biologically active hTrx1 protein in plant chloroplasts. This research opens up possibilities for plant-based production of hTrx1. Considering that this expression host is an edible crop plant, this transplastomic lettuce may be suitable for oral delivery of hTrx1.     

Targeting a nuclear anthranilate synthase alpha-subunit gene to the tobacco plastid genome results in enhanced tryptophan biosynthesis. Return of a gene to its pre-endosymbiotic origin

Anthranilate synthase (AS), the control enzyme of the tryptophan (Trp) biosynthetic pathway, is encoded by nuclear genes, but is transported into the plastids. A tobacco (Nicotiana tabacum) cDNA (ASA2) encoding a feedback-insensitive tobacco AS alpha-subunit was transformed into two different sites of the tobacco plastid genome through site-specific insertion to obtain transplastomic plants with normal phenotype and fertility. A high and uniform level of ASA2 mRNA was observed in the transplastomic plants but not in the wild type. Although the plants with the transgene insertion at ndhF-trnL only expressed one size of the ASA2 mRNA, the plants with the transgene incorporated into the region between accD and open reading frame (ORF) 184 exhibited two species of mRNA, apparently due to readthrough. The transplastomic plants exhibited a higher level of AS alpha-subunit protein and AS enzyme activity that was less sensitive to Trp-feedback inhibition, leading to greatly increased free Trp levels in leaves and total Trp levels in seeds. Resistance to an AS inhibitor, 5-methyl-Trp, was found during seed germination and in suspension cultures of the transplastomic plants. The resistance to the selection agent spectinomycin and to 5-methyl-Trp was transmitted maternally. These results demonstrate the feasibility of modifying the biosynthetic pathways of important metabolites through transformation of the plastid genome by relocating a native gene from the nucleus to the plastid genome. Very high and uniform levels of gene expression can be observed in different lines, probably due to the identical insertion sites, in contrast to nuclear transformation where random insertions occur.     

Chloroplast molecular farming: efficient production of a thermostable xylanase by Nicotiana tabacum plants and long-term conservation of the recombinant enzyme

The high cost of recombinant enzymes for the production of biofuel from ligno-cellulosic biomass is a crucial factor affecting the economic sustainability of the process. The use of plants as biofactories for the production of the suitable recombinant enzymes might be an alternative to microbial fermentation. In the case of enzyme accumulation in chloroplasts, it is fundamental to focus on the issue of full photosynthetic efficiency of transplastomic plants in the field where they might be exposed to abiotic stress such as high light intensity and high temperature. Xylanases (EC 3.2.1.8), a group of enzymes that hydrolyse linear polysaccharides of beta-1,4-xylan into xylose, find an application in the biofuel industry favouring biomass saccharification along with other cell-wall degrading enzymes. In the present study, we analysed how a high level of accumulation of a thermostable xylanase in tobacco chloroplasts does not impact on photosynthetic performance of transplastomic plants grown outdoors. The recombinant enzyme was found to be stable during plant development, ex planta and after long-term storage.     

Immunogenicity of EIT chimeric protein expressed in transplastomic tobacco plants towards development of an oral vaccine against Escherichia coli O157:H7

Chloroplast genetic engineering offers an opportunity for high level expression and cost-effective recombinant protein production. Escherichia coli O157:H7 is one of the most important zoonotic pathogens causing hemorrhagic colitis (HC) and the life-threatening hemolytic-uremic syndrome in humans worldwide. The occurrence of zoonotic E. coli O157:H7 outbreaks in recent years has led to increased efforts in the development of safe and cost-effective immunogenic antigens against E. coli O157:H7. EspA and Tir/Intimin proteins are the important virulence factors which are encoded by the LEE locus of enterohemorrhagic E. coli. In this study, we hypothesized that the high level expression of the chimeric form of these effectors in chloroplasts and using tobacco transplastomic plants as an oral delivery system for the development of an edible-base vaccine would induce an immune response for the prevention of E. coli 0157:H7 attachment and colonization in animal model mice. The prokaryotic codon-optimized EIT protein was expressed in plastid genome via chloroplast transformation. Putative transplastomic plants were analyzed by PCR, and Southern blot analysis confirming chloroplast integration and homoplasmy in the T1 progeny. Immunoblotting and ELISA assays demonstrated that the EIT protein was expressed in chloroplasts and accumulated up to 1.4 % of total soluble protein in leaf tissue. In mice orally immunized with transplastomic tobacco plant leaves, high immunological responses (IgG and IgA specific antibodies) were detected in serum and feces. Finally, the challenging assay with E. coli O157:H7 in immunized mice showed reduced bacterial shedding.     

Contained and high-level production of recombinant protein in plant chloroplasts using a temporary immersion bioreactor

Chloroplast transformation is a promising approach for the commercial production of recombinant proteins in plants. However, gene containment still remains an issue for the large-scale cultivation of transplastomic plants in the field. Here, we have evaluated the potential of using tobacco transplastomic cell suspensions for the fully contained production of a modified form of the green fluorescent protein (GFP+) and, a vaccine antigen, fragment C of tetanus toxin (TetC). Expression of these proteins in cell suspension cultures (and calli) was much less than in leaves, reaching 0.5%-1.5% of total soluble protein (TSP), but still produced 2.4-7.2 mg/L of liquid culture. Much better expression levels were achieved with a novel protein production platform in which transgenic cell suspension cultures were placed in a temporary immersion bioreactor in the presence of Thidiazuron to initiate shoot formation. GFP+ yield reached 660 mg/L of bioreactor (33% TSP), and TetC accumulated to about 95 mg/L (8% TSP). This new production platform, combining the rapid generation of transplastomic cell suspension cultures and the use of temporary immersion bioreactors, is a promising route for the fully contained low-cost production of recombinant proteins in chloroplasts.     

Stable expression of a fungal laccase protein using transplastomic tobacco

Laccase catalyzes the oxidation of various phenolic compounds that can be used in a wide range of industrial applications such as waste detoxification and the textile industry. In the present study, we generated transplastomic tobacco plants to develop a reliable commercial source of laccase production. The stability of the laccase protein in the transgenic plants was increased by using the enhancer sequence from green fluorescent protein, resulting in three independent lines with high levels of laccase accumulation (up to 2?% of total protein); significant laccase activity, however, was not detected. Interestingly, the transplastomic lines showed slightly retarded vegetative growth, with a light green leaf color in comparison with the control, which may be attributable to copper deficiency induced by ligand chelation by abundantly produced laccase. These results suggest that the tobacco chloroplast is an efficient system for the mass production of laccase protein, but further studies are needed to obtain active enzyme.     

SARS亚基疫苗-突刺蛋白受体结合区RBD在烟草叶绿体中的高效表达

叶绿体表达系统为植物源重组药用蛋白和亚基疫苗的生产提供了一个有效的途径。为验证SARS亚基疫苗在叶绿体中表达的可行性,以及为植物源SARS亚基疫苗的生产提供一套高效、低成本的技术平台,本研究将人工优化合成的SARS-CoV突刺蛋白(S蛋白)受体结合区序列RBD与载体分子CTB融合基因导入烟草叶绿体基因组中。PCR和Southern杂交分析表明,外源融合基因已整合到烟草叶绿体基因组中,并获得同质化。Western杂交分析表明,重组融合蛋白CTB-RBD在叶绿体转基因烟草中获得表达,且主要以可溶性单体形式存在。ELISA分析表明,在不同生长阶段、不同生长部位和不同时间点烟草叶片中,重组融合蛋白CTB-RBD的表达水平呈现明显的变化。重组蛋白在成熟叶片中的表达水平最高可以达到10.2%TSP。本研究通过SARS亚基疫苗RBD在烟草叶绿体中的高效表达,有望为植物源SARS亚基疫苗的生产以及SARS血清抗体的检测提供一个有效的技术平台。     

Plastid expression of a double-pentameric vaccine candidate containing human papillomavirus-16 L1 antigen fused with LTB as adjuvant: transplastomic plants show pleiotropic phenotypes

Human papillomavirus (HPV) causes cervical cancer in women worldwide, which is currently prevented by vaccines based on virus-like particles (VLPs). However, these vaccines have certain limitations in their availability to developing countries, largely due to elevated costs. Concerning the highest burden of disease in resource-poor countries, development of an improved mucosal and cost-effective vaccine is a necessity. As an alternative to VLPs, capsomeres have been shown to be highly immunogenic and can be used as vaccine candidate. Furthermore, coupling of an adjuvant like Escherichia coli heat-labile enterotoxin subunit B (LTB) to an antigen can increase its immunogenicity and reduce the costs related to separate co-administration of adjuvants. Our study demonstrates the expression of two pentameric proteins: the modified HPV-16 L1 (L1_2xCysM) and LTB as a fusion protein in tobacco chloroplasts. Homoplasmy of the transplastomic plants was confirmed by Southern blotting. Western blot analysis showed that the LTB-L1 fusion protein was properly expressed in the plastids and the recombinant protein was estimated to accumulate up to 2% of total soluble protein. Proper folding and display of conformational epitopes for both LTB and L1 in the fusion protein was confirmed by GM1-ganglioside binding assay and antigen capture ELISA, respectively. However, all transplastomic lines showed chlorosis, male sterility and growth retardation, which persisted in the ensuing four generations studied. Nevertheless, plants reached maturity and produced seeds by pollination with wild-type plants. Taken together, these results pave the way for the possible development of a low-cost adjuvant-coupled vaccine with potentially improved immunogenicity against cervical cancer.     

Expression of an Escherichia coli antigenic fusion protein comprising the heat labile toxin B subunit and the heat stable toxin, and its assembly as a functional oligomer in transplastomic tobacco plants

Enterotoxigenic Escherichia coli (ETEC) strains are important pathogens in developing countries. Some vaccine formulations containing the heat labile toxin B subunit (LTB) have been used in clinical trials; however, the induction of neutralizing antibodies against the heat-stable toxin (ST), a poor immunogenic peptide, is necessary, as most ETEC strains can produce both toxins. In this study, a plant optimized synthetic gene encoding for the LTB-ST fusion protein has been introduced into plastids of tobacco leaf tissues, using biolistic microprojectile bombardment, in an effort to develop a single plant-based candidate vaccine against both toxins. Transplastomic tobacco plants carrying the LTB-ST transgene have been recovered. Transgene insertion into the plastid was confirmed by both PCR and Southern blot analysis. GM1-ELISA revealed that the LTB-ST fusion protein retained its oligomeric structure, and displayed antigenic determinants for both LTB and ST. Western blot analysis, using LTB antisera, confirmed the presence of a 17-KDa protein in transplastomic lines, with the correct antigenicity of the fusion protein. Expression levels of this fusion protein in different lines reached up to 2.3% total soluble protein. Oral immunization of mice with freeze-dried transplastomic tobacco leaves led to the induction of both serum and mucosal LTB-ST specific antibodies. Following cholera toxin challenge, a decrease of intestinal fluid accumulation was observed in mice immunized with LTB-ST-containing tobacco. These findings suggest that tobacco plants expressing LTB-ST could serve as a plant-based candidate vaccine model providing broad-spectrum protection against ETEC-induced diarrhoeal disease.