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1.  Assessment of Cultivation Factors that Affect Biomass and Geraniol Production in Transgenic Tobacco Cell Suspension Cultures 
PLoS ONE  2014;9(8):e104620.
A large-scale statistical experimental design was used to determine essential cultivation parameters that affect biomass accumulation and geraniol production in transgenic tobacco (Nicotiana tabacum cv. Samsun NN) cell suspension cultures. The carbohydrate source played a major role in determining the geraniol yield and factors such as filling volume, inoculum size and light were less important. Sucrose, filling volume and inoculum size had a positive effect on geraniol yield by boosting growth of plant cell cultures whereas illumination of the cultures stimulated the geraniol biosynthesis. We also found that the carbohydrates sucrose and mannitol showed polarizing effects on biomass and geraniol accumulation. Factors such as shaking frequency, the presence of conditioned medium and solubilizers had minor influence on both plant cell growth and geraniol content. When cells were cultivated under the screened conditions for all the investigated factors, the cultures produced ∼5.2 mg/l geraniol after 12 days of cultivation in shaking flasks which is comparable to the yield obtained in microbial expression systems. Our data suggest that industrial experimental designs based on orthogonal arrays are suitable for the selection of initial cultivation parameters prior to the essential medium optimization steps. Such designs are particularly beneficial in the early optimization steps when many factors must be screened, increasing the statistical power of the experiments without increasing the demand on time and resources.
doi:10.1371/journal.pone.0104620
PMCID: PMC4130582  PMID: 25117009
2.  Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system 
BMC Biotechnology  2014;14:37.
Background
Cell-free protein synthesis is a rapid and efficient method for the production of recombinant proteins. Usage of prokaryotic cell-free extracts often leads to non-functional proteins. Eukaryotic counterparts such as wheat germ extract (WGE) and rabbit reticulocyte lysate (RLL) may improve solubility and promote the correct folding of eukaryotic multi-domain proteins that are difficult to express in bacteria. However, the preparation of WGEs is complex and time-consuming, whereas RLLs suffer from low yields. Here we report the development of a novel cell-free system based on tobacco Bright Yellow 2 (BY-2) cells harvested in the exponential growth phase.
Results
The highly-productive BY-2 lysate (BYL) can be prepared quickly within 4–5 h, compared to 4–5 d for WGE. The efficiency of the BYL was tested using three model proteins: enhanced yellow fluorescent protein (eYFP) and two versions of luciferase. The added mRNA was optimized by testing different 5’ and 3’ untranslated regions (UTRs). The protein yield in batch and dialysis reactions using BYL was much higher than that of a commercial Promega WGE preparation, achieving a maximum yield of 80 μg/mL of eYFP and 100 μg/mL of luciferase, compared to only 45 μg/mL of eYFP and 35 μg/mL of luciferase in WGEs. In dialysis reactions, the BYL yielded about 400 μg/mL eYFP, representing up to 50% more of the target protein than the Promega WGE, and equivalent to the amount using 5Prime WGE system.
Conclusions
Due to the high yield and the short preparation time the BYL represents a remarkable improvement over current eukaryotic cell-free systems.
doi:10.1186/1472-6750-14-37
PMCID: PMC4101825  PMID: 24886601
Cell-free protein synthesis; In vitro translation; Nicotiana tabacum cv. BY-2; Wheat germ extract; Protein expression
3.  Comparative Evaluation of Heterologous Production Systems for Recombinant Pulmonary Surfactant Protein D 
Commercial surfactant products derived from animal lungs are used for the treatment of respiratory diseases in premature neonates. These products contain lipids and the hydrophobic surfactant proteins B and C, which help to lower the surface tension in the lungs. Surfactant products are less effective when pulmonary diseases involve inflammatory complications because two hydrophilic surfactant proteins (A and D) are lost during the extraction process, yet surfactant protein D (SP-D) is a component of the innate immune system that helps to reduce lung inflammation. The performance of surfactant products could, therefore, be improved by supplementing them with an additional source of SP-D. Recombinant SP-D (rSP-D) is produced in mammalian cells and bacteria (Escherichia coli), and also experimentally in the yeast Pichia pastoris. Mammalian cells produce full-size SP-D, but the yields are low and the cost of production is high. In contrast, bacteria produce a truncated form of SP-D, which is active in vitro and in vivo, and higher yields can be achieved at a lower cost. We compare the efficiency of production of rSP-D in terms of the total yields achieved in each system and the amount of SP-D needed to meet the global demand for the treatment of pulmonary diseases, using respiratory distress syndrome as a case study.
doi:10.3389/fimmu.2014.00623
PMCID: PMC4259113  PMID: 25538707
biopharmaceuticals; heterologous production platform; pulmonary surfactant; recombinant protein yield; recombinant surfactant protein D; respiratory distress syndrome
4.  Protective Oral Vaccination against Infectious bursal disease virus Using the Major Viral Antigenic Protein VP2 Produced in Pichia pastoris 
PLoS ONE  2013;8(12):e83210.
Infectious bursal disease virus (IBDV) causes economically important immunosuppressive disease in young chickens. The self-assembling capsid protein (VP2) from IBDV strain IR01 was expressed in Pichia pastoris resulting in the formation of homomeric, 23-nm infectious bursal disease subviral particles (IBD-SVPs) with a yield of 76 mg/l before and 38 mg/l after purification. Anti-IBDV antibodies were detected in chickens injected with purified IBD-SVPs or fed with either purified IBD-SVPs or inactivated P. pastoris cells containing IBD-VP2 (cell-encapsulated). Challenge studies using the heterologous classical IBDV strain (MB3) showed that intramuscular vaccination with 20 µg purified IBD-SVPs conferred full protection, achieved complete virus clearance and prevented bursal damage and atrophy, compared with only 40% protection, 0–10% virus clearance accompanied by severe atrophy and substantial bursal damage in mock-vaccinated and challenge controls. The commercial IBDV vaccine also conferred full protection and achieved complete virus clearance, albeit with partial bursal atrophy. Oral administration of 500 µg purified IBD-SVPs with and without adjuvant conferred 100% protection but achieved only 60% virus clearance with adjuvant and none without it. Moderate bursal damage was observed in both cases but the inclusion of adjuvant resulted in bursal atrophy similar to that observed with live-attenuated vaccine and parenteral administration of 20 µg purified IBD-SVPs. The oral administration of 250 mg P. pastoris cells containing IBD-VP2 resulted in 100% protection with adjuvant and 60% without, accompanied by moderate bursal damage and atrophy in both groups, whereas 25 mg P. pastoris cells containing IBD-VP2 resulted in 90–100% protection with moderate bursal lesions and severe atrophy. Finally, the oral delivery of 50 µg purified IBD-SVPs achieved 40–60% protection with severe bursal lesions and atrophy. Both oral and parenteral administration of yeast-derived IBD-VP2 can therefore induce a specific and protective immune response against IBDV without affecting the growth rate of chickens.
doi:10.1371/journal.pone.0083210
PMCID: PMC3869785  PMID: 24376665
5.  Correlation between mass transfer coefficient kLa and relevant operating parameters in cylindrical disposable shaken bioreactors on a bench-to-pilot scale 
Background
Among disposable bioreactor systems, cylindrical orbitally shaken bioreactors show important advantages. They provide a well-defined hydrodynamic flow combined with excellent mixing and oxygen transfer for mammalian and plant cell cultivations. Since there is no known universal correlation between the volumetric mass transfer coefficient for oxygen kLa and relevant operating parameters in such bioreactor systems, the aim of this current study is to experimentally determine a universal kLa correlation.
Results
A Respiration Activity Monitoring System (RAMOS) was used to measure kLa values in cylindrical disposable shaken bioreactors and Buckingham’s π-Theorem was applied to define a dimensionless equation for kLa. In this way, a scale- and volume-independent kLa correlation was developed and validated in bioreactors with volumes from 2 L to 200 L. The final correlation was used to calculate cultivation parameters at different scales to allow a sufficient oxygen supply of tobacco BY-2 cell suspension cultures.
Conclusion
The resulting equation can be universally applied to calculate the mass transfer coefficient for any of seven relevant cultivation parameters such as the reactor diameter, the shaking frequency, the filling volume, the viscosity, the oxygen diffusion coefficient, the gravitational acceleration or the shaking diameter within an accuracy range of +/− 30%. To our knowledge, this is the first kLa correlation that has been defined and validated for the cited bioreactor system on a bench-to-pilot scale.
doi:10.1186/1754-1611-7-28
PMCID: PMC4177207  PMID: 24289110
Dimensional analysis; Maximum oxygen transfer capacity; Orbitally shaken; Plant cell suspension cultures; Single-use; Volumetric mass transfer coefficient
6.  Plant-Based Production of Recombinant Plasmodium Surface Protein Pf38 and Evaluation of its Potential as a Vaccine Candidate 
PLoS ONE  2013;8(11):e79920.
Pf38 is a surface protein of the malarial parasite Plasmodium falciparum. In this study, we produced and purified recombinant Pf38 and a fusion protein composed of red fluorescent protein and Pf38 (RFP-Pf38) using a transient expression system in the plant Nicotiana benthamiana. To our knowledge, this is the first description of the production of recombinant Pf38. To verify the quality of the recombinant Pf38, plasma from semi-immune African donors was used to confirm specific binding to Pf38. ELISA measurements revealed that immune responses to Pf38 in this African subset were comparable to reactivities to AMA-1 and MSP119. Pf38 and RFP-Pf38 were successfully used to immunise mice, although titres from these mice were low (on average 1∶11.000 and 1∶39.000, respectively). In immune fluorescence assays, the purified IgG fraction from the sera of immunised mice recognised Pf38 on the surface of schizonts, gametocytes, macrogametes and zygotes, but not sporozoites. Growth inhibition assays using αPf38 antibodies demonstrated strong inhibition (≥60%) of the growth of blood-stage P. falciparum. The development of zygotes was also effectively inhibited by αPf38 antibodies, as determined by the zygote development assay. Collectively, these results suggest that Pf38 is an interesting candidate for the development of a malaria vaccine.
doi:10.1371/journal.pone.0079920
PMCID: PMC3836784  PMID: 24278216
7.  Tackling Heterogeneity: A Leaf Disc-Based Assay for the High-Throughput Screening of Transient Gene Expression in Tobacco 
PLoS ONE  2012;7(9):e45803.
Transient Agrobacterium-mediated gene expression assays for Nicotiana tabacum (N. tabacum) are frequently used because they facilitate the comparison of multiple expression constructs regarding their capacity for maximum recombinant protein production. However, for three model proteins, we found that recombinant protein accumulation (rpa) was significantly influenced by leaf age and leaf position effects. The ratio between the highest and lowest amount of protein accumulation (max/min ratio) was found to be as high as 11. Therefore, construct-based impacts on the rpa level that are less than 11-fold will be masked by background noise. To address this problem, we developed a leaf disc-based screening assay and infiltration device that allows the rpa level in a whole tobacco plant to be reliably and reproducibly determined. The prototype of the leaf disc infiltration device allows 14 Agrobacterium-mediated infiltration events to be conducted in parallel. As shown for three model proteins, the average max/min rpa ratio was reduced to 1.4 using this method, which allows for a sensitive comparison of different genetic elements affecting recombinant protein expression.
doi:10.1371/journal.pone.0045803
PMCID: PMC3448687  PMID: 23029251
8.  Development of an optimized tetracycline-inducible expression system to increase the accumulation of interleukin-10 in tobacco BY-2 suspension cells 
BMC Biotechnology  2012;12:40.
Background
Plant cell suspension cultures can be used for the production of valuable pharmaceutical and industrial proteins. When the recombinant protein is secreted into the culture medium, restricting expression to a defined growth phase can improve both the quality and quantity of the recovered product by minimizing proteolytic activity. Temporal restriction is also useful for recombinant proteins whose constitutive expression affects cell growth and viability, such as viral interleukin-10 (vIL-10).
Results
We have developed a novel, tetracycline-inducible system suitable for tobacco BY-2 suspension cells which increases the yields of vIL-10. The new system is based on a binary vector that is easier to handle than conventional vectors, contains an enhanced inducible promoter and 5′-UTR to improve yields, and incorporates a constitutively-expressed visible marker gene to allow the rapid and straightforward selection of the most promising transformed clones. Stable transformation of BY-2 cells with this vector, without extensive optimization of the induction conditions, led to a 3.5 fold increase in vIL-10 levels compared to constitutive expression in the same host.
Conclusions
We have developed an effective and straightforward molecular farming platform technology that improves both the quality and the quantity of recombinant proteins produced in plant cells, particularly those whose constitutive expression has a negative impact on plant growth and development. Although we tested the platform using vIL-10 produced in BY-2 cells, it can be applied to other host/product combinations and is also useful for basic research requiring strictly controlled transgene expression.
doi:10.1186/1472-6750-12-40
PMCID: PMC3410776  PMID: 22784336
9.  A membrane-bound matrix-metalloproteinase from Nicotiana tabacum cv. BY-2 is induced by bacterial pathogens 
BMC Plant Biology  2009;9:83.
Background
Plant matrix metalloproteinases (MMP) are conserved proteolytic enzymes found in a wide range of monocotyledonous and dicotyledonous plant species. Acting on the plant extracellular matrix, they play crucial roles in many aspects of plant physiology including growth, development and the response to stresses such as pathogen attack.
Results
We have identified the first tobacco MMP, designated NtMMP1, and have isolated the corresponding cDNA sequence from the tobacco suspension cell line BY-2. The overall domain structure of NtMMP1 is similar to known MMP sequences, although certain features suggest it may be constitutively active rather than dependent on proteolytic processing. The protein appears to be expressed in two forms with different molecular masses, both of which are enzymatically active as determined by casein zymography. Exchanging the catalytic domain of NtMMP1 with green fluorescent protein (GFP) facilitated subcellular localization by confocal laser scanning microscopy, showing the protein is normally inserted into the plasma membrane. The NtMMP1 gene is expressed constitutively at a low level but can be induced by exposure to bacterial pathogens.
Conclusion
Our biochemical analysis of NtMMP1 together with bioinformatic data on the primary sequence indicate that NtMMP1 is a constitutively-active protease. Given its induction in response to bacterial pathogens and its localization in the plasma membrane, we propose a role in pathogen defense at the cell periphery.
doi:10.1186/1471-2229-9-83
PMCID: PMC2715019  PMID: 19563670
10.  Viral and murine interleukin-10 are correctly processed and retain their biological activity when produced in tobacco 
BMC Biotechnology  2009;9:22.
Background
Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in several autoimmune and inflammatory diseases. Oral administration of this cytokine alone, or in combination with disease-associated autoantigens could confer protection form the onset of a specific autoimmune disease through the induction of oral tolerance. Transgenic plants are attractive systems for production of therapeutic proteins because of the ability to do large scale-up at low cost, and the low maintenance requirements. They are highly amenable to oral administration and could become effective delivery systems without extensive protein purification. We investigated the ability of tobacco plants to produce high levels of biologically-active viral and murine IL-10.
Results
Three different subcellular targeting strategies were assessed in transient expression experiments, and stable transgenic tobacco plants were generated with the constructs that yielded the highest accumulation levels by targeting the recombinant proteins to the endoplasmic reticulum. The best yields using this strategy in T1 plants were 10.8 and 37.0 μg/g fresh leaf weight for viral and murine IL-10, respectively. The recombinant proteins were purified from transgenic leaf material and characterized in terms of their N-glycan composition, dimerization and biological activity in in vitro assays. Both molecules formed stable dimers, were able to activate the IL-10 signaling pathway and to induce specific anti-inflammatory responses in mouse J774 macrophage cells.
Conclusion
Tobacco plants are able to correctly process viral and murine IL-10 into biologically active dimers, therefore representing a suitable platform for the production for these cytokines. The accumulation levels obtained are high enough to allow delivery of an immunologically relevant dose of IL-10 in a reasonable amount of leaf material, without extensive purification. This study paves the way to performing feeding studies in mouse models of autoimmune diseases, that will allow the evaluation the immunomodulatory properties and effectiveness of the viral IL-10 in inducing oral tolerance compared to the murine protein.
doi:10.1186/1472-6750-9-22
PMCID: PMC2667500  PMID: 19298643

Results 1-10 (10)