Production of leafy biomass using temporary immersion bioreactors: an alternative platform to express proteins in transplastomic plants with drastic phenotypes

Chloroplast transformation technology is a promising approach for the production of foreign proteins in plants with expression levels of up to 70 % of total soluble protein (TSP) achieved in tobacco. However, expression of foreign protein in the chloroplast can lead to drastic or even lethal effects in transplastomic plants grown in soil, thereby potentially limiting the applicability of this technology. For instance, previous attempts to express the outer surface protein A (OspA) from Borrelia burgdorferi in tobacco chloroplasts led to plant death when expressed at 10 % TSP. We show here that this earlier transplastomic line, as well as a new plant line, OspA:YFP, expressing OspA fused to the yellow fluorescent protein, can be propagated in temporary immersion bioreactors (TIBs) using AlkaBurst? technology to produce leafy biomass that expressed OspA at levels of up to 7.6 % TSP, to give a maximum yield of OspA of about 108 mg/L. Our results show that TIBs provide an alternative method for the production of transplastomic biomass expressing proteins toxic for plants and is a particularly useful approach when ‘absolute’ containment is required.     

Scale‐up of hydrophobin‐assisted recombinant protein production in tobacco BY‐2 suspension cells

Plant suspension cell cultures are emerging as an alternative to mammalian cells for production of complex recombinant proteins. Plant cell cultures provide low production cost, intrinsic safety and adherence to current regulations, but low yields and costly purification technology hinder their commercialization. Fungal hydrophobins have been utilized as fusion tags to improve yields and facilitate efficient low‐cost purification by surfactant‐based aqueous two‐phase separation (ATPS) in plant, fungal and insect cells. In this work, we report the utilization of hydrophobin fusion technology in tobacco bright yellow 2 (BY‐2) suspension cell platform and the establishment of pilot‐scale propagation and downstream processing including first‐step purification by ATPS. Green fluorescent protein‐hydrophobin fusion (GFP‐HFBI) induced the formation of protein bodies in tobacco suspension cells, thus encapsulating the fusion protein into discrete compartments. Cultivation of the BY‐2 suspension cells was scaled up in standard stirred tank bioreactors up to 600 L production volume, with no apparent change in growth kinetics. Subsequently, ATPS was applied to selectively capture the GFP‐HFBI product from crude cell lysate, resulting in threefold concentration, good purity and up to 60% recovery. The ATPS was scaled up to 20 L volume, without loss off efficiency. This study provides the first proof of concept for large‐scale hydrophobin‐assisted production of recombinant proteins in tobacco BY‐2 cell suspensions.     

High-level secretion of functional green fluorescent protein from transgenic tobacco cell cultures: characterization and sensing

Green fluorescent protein (GFP) is useful for studying protein trafficking in plant cells. This utility could potentially be extended to develop an efficient secretory reporter system or to enable on-line monitoring of secretory recombinant protein production in plant cell cultures. Toward this end, the aim of the present study was to: (1) demonstrate and characterize high levels of secretion of fluorescent GFP from transgenic plant cell culture; and (2) examine the utility of GFP fluorescence for monitoring secreted recombinant protein production. In this study we expressed in tobacco cell cultures a secretory GFP construct made by splicing an Arabidopsis basic chitinase signal sequence to GFP. Typical extracellular GFP accumulation was 12 mg/L after 10 to 12 days of culture. The secreted GFP is functional and it accounts for up to 55% of the total GFP expressed. Findings from culture treatments with brefeldin A suggest that GFP is secreted by the cultured tobacco cells via the classical endoplasmic reticulum-Golgi pathway. Over the course of flask cultures, medium fluorescence increased with the secreted GFP concentrations that were determined using either Western blot or enzyme-linked immunoassay. Real-time monitoring of secreted GFP in plant cell cultures by on-line fluorescence detection was verified in bioreactor cultures in which the on-line culture fluorescence signals showed a linear dependency on the secreted GFP concentrations.     

Bioreactor systems for in vitro production of foreign proteins using plant cell cultures

Plant cells have been demonstrated to be an attractive heterologous expression host (using whole plants and in vitro plant cell cultures) for foreign protein production in the past 20years. In recent years in vitro liquid cultures of plant cells in a fully contained bioreactor have become promising alternatives to traditional microbial fermentation and mammalian cell cultures as a foreign protein expression platform, due to the unique features of plant cells as a production host including product safety, cost-effective biomanufacturing, and the capacity for complex protein post-translational modifications. Heterologous proteins such as therapeutics, antibodies, vaccines and enzymes for pharmaceutical and industrial applications have been successfully expressed in plant cell culture-based bioreactor systems including suspended dedifferentiated plant cells, moss, and hairy roots, etc. In this article, the current status and emerging trends of plant cell culture for in vitro production of foreign proteins will be discussed with emphasis on the technological progress that has been made in plant cell culture bioreactor systems.     

Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells

Transient gene expression (TGE) in mammalian cells at the reactor scale is becoming increasingly important for the rapid production of recombinant proteins. We improved a process for transient calcium phosphate-based transfection of HEK293-EBNA cells in a 1-3 L bioreactor volume. Cells were adapted to suspension culture using a commercially available medium (BioWhittaker, Walkersville, MD). Process parameters were optimized using a plasmid reporter vector encoding the enhanced green fluorescent protein (EGFP/CLONTECH, Palo Alto, CA, USA). Using GFP as a marker-protein, we observed by microscopic examination transfection efficiencies between 70-100%. Three different recombinant proteins were synthesized within a timeframe of 7 days from time of transfection to harvest. The first, a human recombinant IgG(1)-type antibody, was secreted into the supernatant of the cell culture and achieved a final concentration of >20 mg/L. An E. coli-derived DNA-binding protein remained intracellular, as expected, but accumulated to such a concentration that the lysate of cells, taken up into the entire culture volume, gave a concentration of 18 mg/L. The third protein, a transmembrane receptor, was expressed at 3-6 x 10(6) molecules/cell.     

Production of biomass and flavonoid of Gynura procumbens (Lour.) Merr shoots culture in temporary immersion system

Gynura procumbens (Lour.) Merris one of medicinal plant which was carried out used as antioxidant, anticancer, anti-inflammatory, hepatoprotective, and antimicrobial. Many strategies were used to increase the production of biomass and valuable compounds. This study was to investigate the variation effect of growth regulators and immersion frequency on production of biomass and flavonoid contained of G. procumbens shoots culture in temporary immersion bioreactor. Stem nodes were used as an explants and induction of shoots were done in solid MS medium supplemented with many kinds of growth regulator. The best treatments were used to produce biomass and flavonoid compounds in temporary immersion bioreactor; there are combination of IAA 2?mg/L and BA 4, 6, 8?mg/L and immersion frequency (5 min each 3?h; 15?min each 12?h). Results showed that the growths of G. procumbens shoots in solid MS medium were influenced by supplementation of growth regulators. MS medium supplemented with single cytokinine (6 mg/L kinetin) and combination of auxin (IAA) and cytokinine (BA) caused increasing of shoots growth. Production of biomass of G. procumbens in temporary immersion bioreactor was achieved in long immersion interval (12?h) and highest flavonoid production was obtained in combination treatment of immersion frequency 15?min each 12?h and MS medium supplemented with IAA 2?mg/L, BA 8?mg/L.     

Expression of a human anti-rabies virus monoclonal antibody in tobacco cell culture

A Nicotiana tabacum cv. Xanthi cell culture was initiated from a transgenic plant expressing a human anti-rabies virus monoclonal antibody. Within 3 months, plant cell suspension cultures were established and recombinant protein expression was examined. The antibody was stably produced during culture growth. ELISA, protein G purification, Western blotting, and neutralization assay confirmed that the antibody was fully processed, with association of light and heavy-chains, and that it was able to bind and neutralize rabies virus. Quantification of antibody production in plant cell suspension culture revealed 30 microg/g of cell dry weight for the highest-producing culture (0.5 mg/L), 3 times higher than from the original transgenic plant. The same production level was observed 3 months after cell culture initiation. Plant cell suspension cultures were successfully grown in a new disposable plastic bioreactor, with a growth rate and production level similar to that of cultures in Erlenmeyer flasks.     

Production of mouse granulocyte-macrophage colony-stimulating factor by gateway technology and transgenic rice cell culture

To establish a production platform for recombinant proteins in rice suspension cells, we first constructed a Gateway-compatible binary T-DNA destination vector. It provided a reliable and effective method for the rapid directional cloning of target genes into plant cells through Agrobacterium-mediated transformation. We used the approach to produce mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) in a rice suspension cell system. The promoter for the αAmy3 amylase gene, which is induced strongly by sugar depletion, drove the expression of mGM-CSF. The resulting recombinant protein was fused with the αAmy3 signal peptide and was secreted into the culture medium. The production of rice-derived mGM-CSF (rmGM-CSF) was scaled up successfully in a 2-L bioreactor, in which the highest yield of rmGM-CSF was 24.6 mg/L. Due to post-translational glycosylation, the molecular weight of rmGM-CSF was larger than that of recombinant mGM-CSF produced in Escherichia coli. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60.     

Bioreactor engineering for recombinant protein production in plant cell suspension cultures

A review of over 15 years of research, development and commercialization of plant cell suspension culture as a bioproduction platform is presented. Plant cell suspension culture production of recombinant products offers a number of advantages over traditional microbial and/or mammalian host systems such as their intrinsic safety, cost-effective bioprocessing, and the capacity for protein post-translational modifications. Recently significant progress has been made in understanding the bottlenecks in recombinant protein expression using plant cells, including advances in plant genetic engineering for efficient transgene expression and minimizing proteolytic degradation or loss of functionality of the product in cell culture medium. In this review article, the aspects of bioreactor design engineering to enable plant cell growth and production of valuable recombinant proteins is discussed, including unique characteristics and requirements of suspended plant cells, properties of recombinant proteins in a heterologous plant expression environment, bioreactor types, design criteria, and optimization strategies that have been successfully used, and examples of industrial applications.     

Production of recombinant proteins in clonal root cultures using episomal expression vectors

We have developed a fully contained system for expressing recombinant proteins that is based on clonal root cultures and episomal expression vectors. Clonal root lines expressing green fluorescent protein (GFP) or human growth hormone were generated from Nicotiana benthamiana leaves infected with the tobacco mosaic virus-based vector 30B after exposure to Agrobacterium rhizogenes. These lines accumulated GFP at over 50 mg per kg fresh tissue, a level that is comparable with other plant production systems in early stage development. Accumulation of both hGH and GFP in the clonal root lines was sustained over a 3-year period, and in the absence of antibiotic selection. This technology shows promise for commercial production of vaccine antigens and therapeutic proteins in contained facilities.     

Serum-free suspensin large-scale transient transfection of CHO cells in WAVE bioreactors

Here, we report the development of a large-scale transient expression platform utilizing Chinese hamster ovary (CHO) cells. The majority of recombinant proteins and antibodies that are produced for preclinical models and clinical trials are expressed in stably transfected CHO cells. A protocol for transient transfection of CHO cells that is rapid, reproducible, and cost-effective would therefore streamline the process from research to development and help avoid any potential host species induced variation in the molecule of interest. CHO cells were adapted to grow in serum-free suspension conditions in spinner flask cultures in a proprietary in-house developed growth medium. In developing this transient transfection protocol, the parameters optimized included the transfection reagent of choice, the cell density at the time of transfection, the plasmid DNA concentration, and the transfection reagent concentration. Using this optimized protocol, we have expressed recombinant proteins, including antibodies, at an expression level of up to 9.4 mg/L. We also report transient transfections from 500 mL working volume (w.v.) up to 20 L w.v. in a WAVE^TM bioreactor. Using this optimized protocol, it is possible to rapidly (within 10 d) produce up to 100 mg of recombinant protein for further study.     

Selectable high‐yield recombinant protein production in human cells using a GFP/YFP nanobody affinity support

Recombinant protein expression systems that produce high yields of pure proteins and multi‐protein complexes are essential to meet the needs of biologists, biochemists, and structural biologists using X‐ray crystallography and cryo‐electron microscopy. An ideal expression system for recombinant human proteins is cultured human cells where the correct translation and chaperone machinery are present. However, compared to bacterial expression systems, human cell cultures present several technical challenges to their use as an expression system. We developed a method that utilizes a YFP fusion‐tag to generate recombinant proteins using suspension‐cultured HEK293F cells. YFP is a dual‐function tag that enables direct visualization and fluorescence‐based selection of high expressing clones for and rapid purification using a high‐stringency, high‐affinity anti‐GFP/YFP nanobody support. We demonstrate the utility of this system by expressing two large human proteins, TOP2α (340 KDa dimer) and a TOP2β catalytic core (260 KDa dimer). This robustly and reproducibly yields >10 mg/L liter of cell culture using transient expression or 2.5 mg/L using stable expression.     

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.     

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。     

Expression of green fluorescent protein in insect larvae and its application for heterologous protein production

Many eukaryotic proteins have been successfully expressed in insect cells infected with a recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). There are, however, disadvantages with this cell-based system when carried out in suspension cultures at high bioreactor volume (e.g., limited oxygen transfer, susceptibility to contamination, high cost). These problems can be avoided by using whole larvae as the "reactors." There are, however, other problems encountered with larvae, one being their inaccessibility for product sampling. To combat this problem, we have investigated the expression of green fluorescent protein (GFP) as a reporter molecule in Trichoplusia ni insect larvae. A high production level of GFPuv (1.58 mg per larva, 26% of total protein) was obtained, enabling the rapid and non-invasive monitoring of GFP. Bright green light was emitted directly from the large opaque carcasses ( approximately 30mm) after illumination with UV light. Based on the green light intensity and a correlation between intensity and GFP mass, we determined the optimal harvest time (c.a. approximately 3 days post-infection). In parallel experiments, we expressed human interleukin-2 (IL-2) from another recombinant baculovirus with an almost identical expression profile. Since both GFP and IL-2 were rapidly degraded by protease activity during the fourth day post-infection (another disadvantage with larvae), we found an accurate determination of harvest time was critical. Correspondingly, our results demonstrated that GFP was an effective on-line marker for expression of heterologous protein in insect larvae. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 239-247, 1997.     

Production of human serum albumin by sugar starvation induced promoter and rice cell culture

Human serum albumin (HSA) is the most widely used clinical serum protein. Currently, commercial HSA can only be obtained from human plasma, due to lack of commercially feasible recombinant protein expression systems. In this study, inducible expression and secretion of HSA by transformed rice suspension cell culture was established. Mature form of HSA was expressed under the control of the sucrose starvation-inducible rice α Amy3 promoter, and secretion of HSA into the culture medium was achieved by using the α Amy3 signal sequence. High concentrations of HSA were secreted into culture medium in a short time (2–4 days) by sucrose depletion after cell concentrations had reached a peak density in culture medium containing sucrose. The recombinant HSA had the same electrophoretic mobility as commercial HSA and was stable and free from apparent proteolysis in the culture medium. In a flask scale culture with repeated sucrose provision-depletion cycles, HSA was stably produced with yields up to 11.5% of total medium proteins or 15 mg/L per cycle after each sucrose provision-depletion cycle. A bubble column type bioreactor was designed for production of HSA. In the bioreactor scale culture, HSA was produced with yields up to 76.4 mg/L 4 days after sucrose depletion. HSA was purified from the culture medium to high purity by a simple purification scheme. Enrichment of HSA in culture medium simplifies downstream purification, minimizes protease degradation, and may reduce production cost. The combination of a DNA construct containing the α Amy3 promoter and signal sequence, and the use of a rice suspension cell culture can provide an effective system for the production of recombinant pharmaceutical proteins.     

Preparation of isotopically labelled recombinant β-defensin for NMR studies

Apoptosis is a major problem in animal cell cultures during production of biopharmaceuticals, such as recombinant proteins or viral vectors. A 293 cell line constitutively expressing vMIA (viral mitochondria-localized inhibitor of apoptosis) was constructed and examined on production of a model recombinant protein, green fluorescent protein (GFP) in the adenovirus-293 expression system, and on production of a model infectious adenoviral vector. vMIA-293 cells were more resistant than the parental 293 cells to apoptosis induced by either oxidative stress, or by adenovirus infection. The yield of GFP produced in vMIA-293 cell cultures was consistently higher (140%) compared to that in the parental cells. vMIA reduced production of adenovirus infectious particles, which was not due to a decline of adenovirus replication, since adenoviral DNA replication rate in vMIA-293 cells was higher than that in the parental cells.In conclusion, introduction of the vMIA gene into the 293 cell line is a promising strategy to improve recombinant protein production in the adenovirus-293 expression system.     

Artemisia judaica L.: micropropagation and antioxidant activity

Artemisia judaica L., an Egyptian medicinal plant used in the treatment of gastrointestinal disorders, was mass-propagated and grown using solid, paper-bridge-support liquid, liquid-flask and bioreactor cultures. The liquid-flask culture using 50 ml MS liquid medium in 250 ml flask yielded significantly greater shoot proliferation than either solid cultures or paper-bridge-support liquid cultures. Increasing flask capacity from 100 to 500 ml improved shoot proliferation and growth. Mass-propagation efficiencies of various bioreactor systems, viz. temporary immersion reactors and bubble column reactors, were also compared. The temporary immersion bioreactor was found to have significant advantages for A. judaica shoot proliferation. The shoot cultures from the temporary immersion reactor formed complete plantlets when subcultured onto a medium containing 1 micromoll(-1) indole-3-butyric acid (IBA), and mature plants were established, acclimatized and thrived in standard greenhouse conditions. Assays of antioxidant activity and total flavonoid content of in vitro and in vivo grown tissues were evaluated as gross parameters of medicinal efficacy. Significantly higher antioxidant activity and flavonoid contents were observed in the tissues of mature greenhouse-grown plants. The efficient in vitro production systems developed in this study provided sterile, consistent tissues for investigation of bioactivity and germplasm conservation of A. judaica.     

High‐yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development

Low‐yield protein production remains the most significant economic hurdle with plant cell culture technology. Fusions of recombinant proteins with hydroxyproline‐O‐glycosylated designer glycopeptide tags have consistently boosted secreted protein yields. This prompted us to study the process development of this technology aiming to achieve productivity levels necessary for commercial viability. We used a tobacco BY‐2 cell culture expressing EGFP as fusion with a glycopeptide tag comprised of 32 repeat of ”Ser‐Pro“ dipeptide, or (SP)₃₂, to study cell growth and protein secretion, culture scale‐up, and establishment of perfusion cultures for continuous production. The BY‐2 cells accumulated low levels of cell biomass (～7.5 g DW/L) in Schenk & Hildebrandt medium, but secreted high yields of (SP)₃₂‐tagged EGFP (125 mg/L). Protein productivity of the cell culture has been stable for 6.0 years. The BY‐2 cells cultured in a 5‐L bioreactor similarly produced high secreted protein yield at 131 mg/L. Successful operation of a cell perfusion culture for 30 days was achieved under the perfusion rate of 0.25 and 0.5 day^?1, generating a protein volumetric productivity of 17.6 and 28.9 mg/day/L, respectively. This research demonstrates the great potential of the designer glycopeptide technology for use in commercial production of valuable proteins with plant cell cultures.     

High levels of stable phytase accumulate in the culture medium of transgenic Medicago truncatula cell suspension cultures

The use of plants for production of recombinant proteins is becoming widely accepted. More recently, plant cell cultures have been proposed as valuable systems for producing a wide range of biologically active proteins. Such systems provide certain advantages over whole plants, but yields are still considered a limitation. In this study we established a Medicago truncatula cell suspension line expressing phytase from Aspergillus niger. Phytase is an N-glycosylated enzyme that breaks down indigestible phytate, resulting in an increased availability of phosphorus and other minerals in monogastric animals and reduced levels of phosphorus output in their manure. Various production systems have previously been used to express heterologous phytase, including several plant species. In this work, remarkable amounts of enzymatically active recombinant phytase were produced and secreted into the culture medium. Recombinant phytase accumulated to at least 25 mg/L and remained stable along the growth curve, and an enriched fraction with high enzymatic activity was easily obtained. We therefore propose M. truncatula cell suspension cultures as a potential system for the production of recombinant proteins. Most importantly, we have shown that, contrary to general belief, it is possible to achieve high levels of a functional recombinant protein in plant cell culture systems.