Tobacco plastidial thioredoxins as modulators of recombinant protein production in transgenic chloroplasts

Thioredoxins (Trxs) are small ubiquitous disulphide proteins widely known to enhance expression and solubility of recombinant proteins in microbial expression systems. Given the common evolutionary heritage of chloroplasts and bacteria, we attempted to analyse whether plastid Trxs could also act as modulators of recombinant protein expression in transgenic chloroplasts. For that purpose, two tobacco Trxs (m and f) with different phylogenetic origins were assessed. Using plastid transformation, we assayed two strategies: the fusion and the co-expression of Trxs with human serum albumin (HSA), which was previously observed to form large protein bodies in tobacco chloroplasts. Our results indicate that both Trxs behave similarly as regards HSA accumulation, although they act differently when fused or co-expressed with HSA. Trxs-HSA fusions markedly increased the final yield of HSA (up to 26% of total protein) when compared with control lines that only expressed HSA; this increase was mainly caused by higher HSA stability of the fused proteins. However, the fusion strategy failed to prevent the formation of protein bodies within chloroplasts. On the other hand, the co-expression constructs gave rise to an absence of large protein bodies although no more soluble HSA was accumulated. In these plants, electron micrographs showed HSA and Trxs co-localization in small protein bodies with fibrillar texture, suggesting a possible influence of Trxs on HSA solubilization. Moreover, the in vitro chaperone activity of Trx m and f was demonstrated, which supports the hypothesis of a direct relationship between Trx presence and HSA aggregates solubilization in plants co-expressing both proteins.     

Improved expression of recombinant plant-made hEGF

Key message

The yield of recombinant hEGF was increased approximately tenfold through a range of optimisations. Further, the recombinant protein was found to have biological activity comparable to commercial hEGF.

Abstract

Human epidermal growth factor (hEGF) is a powerful mitogen that can enhance the healing of a wide range of injuries, including burns, cuts, diabetic ulcers and gastric ulcers. However, despite its clinical value, hEGF is only consistently used for the treatment of chronic diabetic ulcers due to its high cost. In this study, hEGF was transiently expressed in Nicotiana benthamiana plants and targeted to the apoplast, ER and vacuole. Several other approaches were also included in a stepwise fashion to identify the optimal conditions for the expression of recombinant hEGF. Expression was found to be highest in the vacuole, while targeting hEGF to the ER caused a decrease in total soluble protein (TSP). Using a codon optimised sequence was found to increase vacuolar targeted hEGF yield by ~34 %, while it was unable to increase the yield of ER targeted hEGF. The use of the P19 silencing inhibitor was able to further increase expression by over threefold, and using 5-week-old plants significantly increased expression compared to 4- or 6-week-old-plants. The combined effect of these optimisations increased expression tenfold over the initial apoplast targeted construct to an average yield of 6.24 % of TSP. The plant-made hEGF was then shown to be equivalent to commercial E. coli derived hEGF in its ability to promote the proliferation of mouse keratinocytes. This study supports the potential for plants to be used for the commercial production of hEGF, and identifies a potential limitation for the further improvement of recombinant protein yields.     

High-level expression, purification, and crystallization of recombinant rat leukotriene C(4) synthase from the yeast Pichia pastoris

Chemical-enzymatic synthesis of human Epidermal Growth Factor (hEGF) cDNA has been performed, following by cloning into expression vector pTWIN1 (New England Biolabs). The resulting recombinant fusion protein expressed in Escherichia coli consisted of the N-terminal chitin-binding domain, mini-intein Ssp dnaB domain and hEGF polypeptide at the C-terminus. In this construct, mini-intein Ssp dnaB played a role of catalytically active subunit capable under certain conditions of autocatalytic cleavage resulting in separation of the target protein. As the hybrid protein had several cysteins in its sequence-one in chitin-binding domain, one in mini-intein and six in hEGF, it was necessary to work out optimal scheme for refolding and purification of the recombinant hEGF. As a result of this work, two schemes of the recombinant hEGF purification have been developed: according to the first scheme, the recombinant protein with reduced cysteins is bound to the chitin column, the hEGF is cleaved off and eluted, and then refolded to form appropriate cystein bridges. In the second scheme, the entire hybrid protein is first refolded to form disulfide bonds and then loaded to affinity resin; the recombinant hEGF is cleaved off and eluted in its native state. In spite of the fact that the first scheme is more common and suitable for a variety of recombinant proteins, in case of recombinant hEGF, the second scheme proved to be more productive and cost-effective.     

A novel chloroplast transformation vector compatible with the Gateway® recombination cloning technology

To analyze the suitability of Gateway^® vectors for transformation of chloroplasts, we converted a standard plastid transformation vector into a Gateway^® destination vector containing the necessary recombination sites attR1 and attR2. Insertion of the green fluorescent protein (GFP) coding sequence with associated T7g10 ribosome binding site into this destination vector created the expression vector for transformation of tobacco chloroplasts with the biolistic method. Correct integration of the transgene into the plastid genome was verified by PCR and the homoplasmic nature of the transformed plants was confirmed by Southern Blot analysis. Expression of the GFP reporter protein was monitored by confocal laser scanning microscopy (CLSM) and quantification by western blot analysis showed a GFP accumulation level of 3 % total soluble protein (TSP). The presented results clearly demonstrate that the Gateway^® recombination sites are compatible with all steps of plastid transformation, from generation of transplastomic plants to expression of GFP. This is the first report of a plastid transformation vector made by the Gateway^® recombinant cloning technology, which proves the suitability of this system for use in chloroplasts.     

Purification of recombinant human epidermal growth factor secreted from the methylotrophic yeast Hansenula polymorpha.

The gene encoding human epidermal growth factor (hEGF) was expressed as a fusion protein with the Saccharomyces cerevisiae-derived prepro alpha-factor leader in the methylotrophic yeast Hansenula polymorpha. The recombinant hEGF(1-53), when secreted by H. polymorpha, rapidly cleaved to hEGF(1-52) by carboxy-terminal proteolysis, resulting in the accumulation of C-terminal-truncated hEGF(1-52) in the culture medium. To solve this problem, we constructed a H. polymorpha mutant in which the KEX1 gene coding for carboxypeptidase ysc(alpha) was disrupted. The extent of C-terminal proteolysis of hEGF was significantly reduced when this kex1 disruptant was used as a host strain. After 24 h of shake-flask culture, most of the hEGF secreted by the kex1 disruptant remained intact, whereas more than 90% of the hEGF secreted by the wild-type was C-terminally cleaved. The recombinant hEGF was purified to >98% purity by two sequential steps of preparative scale anion exchange chromatography and reverse-phase HPLC. The authenticity of purified hEGF was confirmed by HPLC, N-terminal amino acid sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analyses.     

High-level expression and purification of human epidermal growth factor with SUMO fusion in Escherichia coli

Human epidermal growth factor (hEGF) can stimulate the division of various cell types and has potential clinical applications. However, the high expression of active hEGF in Escherichia coli has not been successful, as the protein contains three intra-molecular disulfide bonds that are difficult to form correctly in the bacterial intracellular environment. To solve this problem, we fused the hEGF gene with a small ubiquitin-related modifier gene (SUMO) by synthesizing an artificial SUMO-hEGF fusion gene that was highly expressed in Origami (DE3) strain. The optimal expression level of the soluble fusion protein, SUMO-hEGF, was up to 38.9% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease to obtain the native hEGF, which was further purified by Ni-NTA affinity chromatography. The result of the reverse-phase HPLC showed that the purity of the recombinant cleaved hEGF was greater than 98%. The primary structure of the purified hEGF was confirmed by N-terminal amino acid sequencing and MALDI-TOF mass spectroscopy analysis. Using the method of methylthiazoletetrazolium, the mitogenic activity on Balb/c 3T3 cells of the purified hEGF was comparable to that of commercial hEGF.     

转基因迷你番茄及其对酒精引起的胃伤害的保护作用

表皮生长因子(EGF)具有促进多种细胞增殖的作用, 尤其在维持消化道黏膜完整和促进消化道溃疡愈合方面作用巨大。以前的研究多集中在细菌和酵母中表达, 本研究试图以番茄作为生物反应器生产重组人 EGF, 使之成本降低, 应用方便。依据人 EGF 的基因序列, 设计合成了番茄密码子偏爱的人 EGF 基因, 并将其构建到植物表达载体 pCAMBIA2300 中, 通过农杆菌介导得到了含有人 EGF 基因的转基因番茄。放射免疫法检测到每克鲜重果实中的表达量达 3.48 ± 1.01 ng。将果汁灌喂小白鼠 15 天(相当于每鼠每天喂服 24 ng rhEGF)能显著抵抗酒精引起的胃溃疡形成, 溃疡指数由 42.20 ± 18.13 下降为 16.25 ± 9.57。     

High-yield expression of a viral peptide animal vaccine in transgenic tobacco chloroplasts

The 2L21 peptide, which confers protection to dogs against challenge with virulent canine parvovirus (CPV), was expressed in tobacco chloroplasts as a C-terminal translational fusion with the cholera toxin B subunit (CTB) or the green fluorescent protein (GFP). Expression of recombinant proteins was dependent on plant age. A very high-yield production was achieved in mature plants at the time of full flowering (310 mg CTB-2L21 protein per plant). Both young and senescent plants accumulated lower amounts of recombinant proteins than mature plants. This shows the importance of the time of harvest when scaling up the process. The maximum level of CTB-2L21 was 7.49 mg/g fresh weight (equivalent to 31.1% of total soluble protein, TSP) and that of GFP-2L21 was 5.96 mg/g fresh weight (equivalent to 22.6% of TSP). The 2L21 inserted epitope could be detected with a CPV-neutralizing monoclonal antibody, indicating that the epitope is correctly presented at the C-terminus of the fusion proteins. The resulting chimera CTB-2L21 protein retained pentamerization and G(M1)-ganglioside binding characteristics of the native CTB and induced antibodies able to recognize VP2 protein from CPV. To our knowledge, this is the first report of an animal vaccine epitope expression in transgenic chloroplasts. The high expression of antigens in chloroplasts would reduce the amount of plant material required for vaccination (approximately 100 mg for a dose of 500 microg antigen) and would permit encapsulation of freeze-dried material or pill formation.     

Molecular design of photosynthesis-elevated chloroplasts for mass accumulation of a foreign protein

In order to increase production of a useful protein by the chloroplast transformation technique, it seems to be necessary to determine the upper limit for the accumulation of a biologically active foreign protein in chloroplasts and then improve photosynthetic capacity and plant productivity. Here we show that the stromal fractions of tobacco chloroplasts could accommodate an additional 200-260 mg ml(-1) of green fluorescent protein in the stroma without any inhibition of gas exchange under various light intensity and growth conditions. The minimum amount of fructose-1,6-/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) limiting photosynthesis was then calculated. Analyses of the photosynthetic parameters and the metabolites of transformants into which FBP/SBPase was introduced with various types of promoter (PpsbA, Prrn, Prps2 and Prps12) indicated that a 2- to 3-fold increase in levels of FBPase and SBPase activity is sufficient to increase the final amount of dry matter by up to 1.8-fold relative to the wild-type plants. Their increases were equivalent to an increase of <1 mg ml(-1) of the FBP/SBPase protein in chloroplasts and were calculated to represent <1% of the protein accumulated via chloroplast transformation. Consequently, >99% of the additional 200-260 mg ml(-1) of protein expressed in the chloroplasts could be used for the production of useful proteins in the photosynthesis-elevated transplastomic plants having FBP/SBPase.     

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.     

Secretion of human epidermal growth factor by Corynebacterium glutamicum

AIMS: To examine the secretion of human epidermal growth factor (hEGF) by Corynebacterium glutamicum. METHODS AND RESULTS: We recently showed that a novel protein-secretion system in C. glutamicum could produce Streptomyces mobaraensis transglutaminase. In the present study, the industrially important protein hEGF was secreted into the culture medium in a fully active form by C. glutamicum and accumulated at a rate of up to 156 mg l(-1) day(-1). CONCLUSIONS: These results demonstrated that the hEGF protein could be secreted in an active form by C. glutamicum. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data confirmed that the pharmaceutically important human protein hEGF could be efficiently secreted in an active form by the C. glutamicum protein-expression system. Moreover, we demonstrated that this bacterium has potential as a host for the industrial-scale production of human proteins.     

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 the truncated tissue plasminogen activator (K2S) gene in tobacco chloroplast

As because the plant plastid genome is highly polyploid, the transformation of chloroplasts permits the introduction of thousands of copies of foreign genes per plant cell and generates extraordinarily high levels of recombinant protein. Human tissue-type plasminogen activator is one of the most important pharmaceutical proteins involved in the breakdown of blood clots in brain and heart blood vessels. We report the introduction and expression of the truncated human tissue plasminogen activator (K2S) gene in tobacco chloroplasts. The K2S-containing vector pKCZK2S was successfully transferred to tobacco plastomes using the biolistic delivery procedure. Transplastomic plants were selected on RMOP medium containing spectinomycin (500 mg/l). In order to achieve homoplasmy, several rounds of selection and regeneration were performed. The presence, site-specific integration, homoplasmy, expression and activity assay of the transgene were confirmed in the transplastomic plants by PCR, Southern-blot, RT-PCR, SDS-PAGE, ELISA, Dot-blot, Western-blot and zymography analysis. Our results show that the tissue plasminogen activator (K2S form) protein to be expressed in tobacco chloroplasts in active form.     

高等植物叶绿体表达重组蛋白研究进展

近年来,基因工程技术发展迅速,许多重组蛋白得以表达。其中利用植物生物反应器表达特异药物蛋白为人类一些重要疾病的预防和治疗提供了新途径。植物叶绿体遗传转化和表达系统成为目前植物生物反应器的研究热点。因结构和遗传上的特殊性,高等植物叶绿体在重组蛋白表达方面具有独特优势,外源基因表达量高、定点整合,而且叶绿体母系遗传特性保证了生物安全性。很多重要药用蛋白质在植物叶绿体中表达成功。烟草作为高等植物叶绿体转化模式植物,在疫苗抗原、抗体等药物蛋白和其他重要重组蛋白表达方面取得显著进展。高等植物叶绿体遗传转化也为叶绿体基因的表达和调控机制的研究提供新的技术和方法。文中从叶绿体遗传转化原理、载体构建、重组蛋白和重要药物蛋白在叶绿体中的表达以及重组蛋白表达对植物代谢和性状影响等多个角度,对高等植物叶绿体遗传转化体系研究的新进展进行了综述,以期为叶绿体表达平台的开发和重要药用蛋白质的表达提供新思路。     

Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit

Transgenic chloroplasts offer unique advantages in plant biotechnology, including high-level foreign protein expression, absence of epigenetic effects, and gene containment due to the lack of transgene transmission through pollen. However, broad application of plastid genome engineering in biotechnology has been largely hampered by both the lack of chloroplast transformation systems for major crop plants and the usually low plastid gene expression levels in nongreen tissues such as fruits, tubers, and other storage organs. Here we describe the development of a plastid transformation system for tomato, Lycopersicon esculentum. This is the first report on the generation of fertile transplastomic plants in a food crop with an edible fruit. We show that chromoplasts in the tomato fruit express the transgene to approximately 50% of the expression levels in leaf chloroplasts. Given the generally very high foreign protein accumulation rates that can be achieved in transgenic chloroplasts (>40% of the total soluble protein), this system paves the way to efficient production of edible vaccines, pharmaceuticals, and antibodies in tomato.     

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.     

High-level expression of the HIV-1 Pr55gag polyprotein in transgenic tobacco chloroplasts

Plants have been recognized as a promising production platform for recombinant pharmaceutical proteins. The human immunodeficiency virus Gag (Pr55^gag) structural polyprotein precursor is a prime candidate for developing a HIV-1 vaccine, but, so far, has been expressed at very low level in plants. The aim of this study was to investigate factors potentially involved in Pr55^gag expression and increase protein yield in plant cells. In transient expression experiments in various subcellular compartments, the native Pr55^gag sequence could be expressed only in the chloroplast. Experiments with truncated subunits suggested a negative role of the 5′-end on the expression of the full gene in the cytosol. Stable transgenic plants were produced in tobacco by Agrobacterium-mediated nuclear transformation with protein targeted to plastids, and biolistic-mediated plastid transformation. Compared to the nuclear genome, the integration and expression of the gag transgene in the plastome resulted in significantly higher protein accumulation levels (up to 7–8% TSP, equivalent to 312–363 mg/kg FW). In transplastomic plants, a 25-fold higher protein accumulation was obtained by translationally fusing the Pr55^gag polyprotein to the N-terminus of the plastid photosynthetic RbcL protein. In chloroplasts, the Pr55^gag polyprotein was processed in a pattern similar to that achieved by the viral protease, the processing being more extended in older leaves of mature plants. The Gag proteins produced in transgenic plastids were able to assemble into particles resembling VLPs produced in baculovirus/insect cells and E. coli systems. These results indicate that plastid transformation is a promising tool for HIV antigen manufacturing in plant cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. IGV publication no. 330     

Recombination and Heterologous Expression of Aliophycocyanin Gene in the Chloroplast of Chlamydomonas reinhardtii

Heterogeneous expression of multiple genes in the nucleus of transgenic plants requires theintroduction of an individual gene and the subsequent backcross to reconstitute multi-subunit proteins ormetabolic pathways.In order to accomplish the expression of multiple genes in a single transformationevent,we inserted both large and small subunits of allophycocyanin gene (apcA and apcB) into Chlamydomonasreinhardtii chloroplast expression vector,resulting in papc-S.The constructed vector was then introducedinto the chloroplast of C.reinhardtii by micro-particle bombardment.Polymerase chain reaction and Southernblot analysis revealed that the two genes had integrated into the chloroplast genome.Western blot andenzyme-linked immunosorbent assay showed that the two genes from the prokaryotic cyanobacteria couldbe correctly expressed in the chloroplasts of C.reinhardtii.The expressed foreign protein in transformantsaccounted for about 2%-3% of total soluble proteins.These findings pave the way to the reconstitution ofmulti-subunit proteins or metabolic pathways in transgenic C.reinhardtii chloroplasts in a single transformationevent.     

Recombination and heterologous expression of allophycocyanin gene in the chloroplast of Chlamydomonas reinhardtii

Heterogeneous expression of multiple genes in the nucleus of transgenic plants requires the introduction of an individual gene and the subsequent backcross to reconstitute multi-subunit proteins or metabolic pathways. In order to accomplish the expression of multiple genes in a single transformation event, we inserted both large and small subunits of allophycocyanin gene （apcA and apcB） into Chlamydomonas reinhardtii chloroplast expression vector, resulting in papc-S. The constructed vector was then introduced into the chloroplast of C. reinhardtii by micro-particle bombardment. Polymerase chain reaction and Southern blot analysis revealed that the two genes had integrated into the chloroplast genome. Western blot and enzyme-linked immunosorbent assay showed that the two genes from the prokaryotic cyanobacteria could be correctly expressed in the chloroplasts of C. reinhardtii. The expressed foreign protein in transformants accounted for about 2%-3% of total soluble proteins. These findings pave the way to the reconstitution of multi-subunit proteins or metabolic pathways in transgenic C. reinhardtii chloroplasts in a single transformation event.