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Comprehensive characterization of two different Nicotiana tabacum cell lines leads to doubled GFP and HA protein production by media optimization
Journal of Bioscience and Bioengineering VOL. 113 No. 2, 242 – 248, 2012 www.elsevier.com/locate/jbiosc
Comprehensive characterization of two different Nicotiana tabacum cell lines leads to doubled GFP and HA protein production by media optimization David A. Ullisch, 1 Christina A. Müller, 1 Sabrina Maibaum, 1 Janina Kirchhoff, 2 Andreas Schiermeyer, 2 Stefan Schillberg, 2 Jean L. Roberts, 3 Wiltrud Treffenfeldt, 3 and Jochen Büchs 1,⁎ RWTH Aachen University, AVT – Biochemical Engineering, Worringer Weg 1, 52056 Aachen, Germany, 1 Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Department Plant Biotechnology, Forckenbeckstrasse 6, 52074 Aachen, Germany, 2 and Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA 3 Received 23 May 2011; accepted 29 September 2011 Available online 4 November 2011
For over two decades, plant cell cultures have been a promising research platform to express recombinant and therapeutic proteins such as hormones, growth factors, full-size antibodies and antigens. Chosen as a good host for manufacturing recombinant proteins, the Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cell line has been studied in shake flasks by offline analysis of only a few growth parameters. The objective of this study is to comprehensively characterize the growth of a transgenic BY-2 cell line and to investigate the expression profile of the model protein GFP. Based on the correlations between nutrient consumption, cell growth and product formation, the intention is to improve the standard MS-medium. Hereby, multiple growth parameters were analyzed offline and online by using a respiration activity monitoring system (RAMOS). A reproducibly observed shift of the oxygen transfer rate (OTR) could be identified to indicate ammonium depletion in the medium. Concurrent with this ammonium depletion, the total protein concentration began to decrease. After the MS-medium was improved, the GFP concentration nearly doubled. When this improved ammonium enriched medium was applied to another transgenic tobacco cell line similar improvements to the amount of the glycoprotein influenza hemagglutinin (HA) produced by Nicotiana tabacum NT-1 cells could be achieved. Ultimately, this combined offline and online analysis can be successfully used for further cell line characterization and media optimization to improve growth and boost target product formation. © 2011, The Society for Biotechnology, Japan. All rights reserved. [Keywords: BY-2 cells; Influenza hemagglutinin; Media optimization; Nitrogen source; Oxygen transfer rate; Plant cell culture; respiration activity monitoring system (RAMOS); Shake flasks]
The number of recombinant proteins produced in plants has steadily increased since the first recombinant protein was expressed in a plant cell culture in the early nineties (1). After this breakthrough, different transgenic plant cell suspension cultures have been used producing recombinant proteins including rice (2), soybeans (3) and tomatoes (4). The production of recombinant proteins from transgenic suspension cultures offers many advantages over that from intact transgenic plants including a better process control and fast generation of transgenic cell lines (5). Another advantage is, that the target protein can be potentially secreted to the culture broth which facilitates the separation and purification. In addition plant cell cultures are not exposed to agrochemicals and variable cultivation conditions due to changes of weather and other environmental conditions (6) and moreover plant cells have their well-documented ability to perform post-translational modifications (7). The tobacco cell line Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2), first isolated by Kato et al. (8) , was chosen as a preferred host cell line for ⁎ Corresponding author. Tel.: + 49 241 8023569; fax: + 49 241 8022570. E-mail address: [email protected] (J. Büchs). Abbreviations: BY-2, Nicotiana tabacum L. cv. Bright Yellow 2; NT-1, Nicotiana tabacum NT-1; OTR, oxygen transfer rate; PBS, phosphate buffered saline; PBST, phosphate buffered saline containing 0.05% (w/v) Tween 20; RAMOS, respiration activity monitoring system.
plant suspension cultures, since this cell line is well characterized — showing high growth rates and high cell synchrony (9). According to Combettes et al. (10) and David and Perrot-Rechenmann (11) this cell line is the model cell line for studying of plant cell cycles and investigating the cellular biology of plants. Up to now, a number of various recombinant proteins, such as antibody fragments (12,13), fullsize antibodies (14,15), enzymes (16–18) and even mature immunologically active allergens (19) have been produced in BY-2 cells. Species-specific nutrient requirements have been described for a wide range of different plant cell cultures such as rice (20), soybeans (21) and tobacco (22). Therefore, different basal salt media have been empirically developed solely by trial and error, and subsequently dose–response experiments have been conducted. Nitrogen – which is the macronutrient with the highest concentration in media – is a constituent of both nucleic acids and proteins and is thus essential to plant life. Nitrogen plays a pivotal role in plant cell metabolism and is directly connected to amino acid and protein biosynthesis (23). Most commonly used nitrogen sources in media for cultivating plant cells are nitrate, ammonium, urea and amino acids. The MS-medium (22) consists of a mixture of two nitrogen sources in a defined ratio, namely nitrate and ammonium. The ratio of nitrate to ammonium ions plays an important role in the metabolism of different plants and has been already discussed in literature (24,25). The assimilation of
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ammonium and nitrate ions is shown in Fig. 1. Ammonium is a readily metabolizable source of nitrogen and is directly transformed into glutamine and glutamic acid by glutamine synthetase (GS) and glutamate synthase (GOGAT) (26). However, cells cannot grow in a medium with ammonium as the sole nitrogen source (27). Explanations for that are the latent toxicity induced by an excess of ammonium ions and the need to control the pH (28). Without control, the pH of the media containing ammonium as the sole nitrogen source falls rapidly to a point where no cell growth is possible (29). But if Krebs cycle acids such as citrate, malate, fumarate or succinate are added to the MSmedium plant cell growth can be observed (30). Nitrate, in contrast, has to be reduced to ammonium before being utilized (Fig. 1). Reduction of ammonium to nitrate is a two-step reaction catalyzed by the nitrate reductase (NR) and by the nitrite reductase (NiR). This reduction is performed by the cell at the expense of 4 mol of NAD(P)H per mol of nitrate (31). On the one hand the presence of nitrate induces both enzymes (32,33); on the other hand ammonium and amino acids can inhibit the activity of these enzymes (34,35). Thus, the pathway is regulated by its substrates and products. Some plants can grow in a medium with nitrate as sole nitrogen source, but BY-2 cell growth is better when the medium contains a mixture of nitrate and ammonium (30). Urea, however, can be used as a sole nitrogen source, but the growth is slower than that in a mixture of ammonium and nitrate (36). Up to now, individual studies have only handled one or two parameters (i.e., biomass or osmolality or conductivity) for studying cell growth. Such limited studies, however, do not provide a comprehensive insight into BY-2 cell metabolism. Moreover, even though the MS-medium has been optimized for rapid tobacco cell growth (22), it has not been optimized for recombinant protein formation. Thus, the objective of this study is to assay multiple growth parameters both offline and online in order to gain a deeper insight into BY-2 cell metabolism. A further objective is to optimize the standard MS-medium to increase target protein production. MATERIALS AND METHODS Plant cell lines Suspension cultures of the transgenic Nicotiana tabacum L. cv. BY-2 cell line expressing GFP were developed from wild-type strain N. tabacum L. cv. BY-2. This wild-type strain was transformed using Agrobacterium tumefaciens (strain LBA4404) containing the vector pDAB100352. A. tumefaciens harboring pDAB100352 was co-incubated with wild-type N. tabacum BY-2 suspension cultures. The transformed plant cells were selected using the herbicide imazethapyr. The T-DNA of pDAB100352 encodes the expression cassette for ER-retarded (endoplasmatic reticulum) green fluorescent protein (GFP-KDEL) (37) under the control of the Cassava vein mosaic virus (CsVMV) promoter (38). The transgenic N. tabacum NT-1 cell line expressing the glycoprotein influenza hemagglutinin (HA) contains the plasmid previously described (Cardineau, G. A., Manson, H. S., VanEck, J. M., Kirk, D. D., and Walmsley, A. M., US patent 2009/0017065 A1, 2009). The expression of HA was also driven
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by the Cassava vein mosaic virus promoter and the recombinant HA was targeted to the endomembrane system of the plant cell. General cultivation conditions BY-2 suspension cultures (250 ml Erlenmeyer flasks with a filling volume of 50 ml) were maintained in MS-medium (22), supplemented with 30 g/l sucrose, 0.2 g/l KH2PO4, 0.6 mg/l thiamine-HCl and 0.2 mg/l 2.4-D. NT-1 suspension cultures (250 ml Erlenmeyer flasks with a filling volume of 50 ml), however, were maintained in MS-medium, supplemented with 30 g/l sucrose, 274.8 mg/l K2HPO4, 170 mg/l KH2PO4, 1 mg/l thiamine-HCL, 4.44 mg/l 2.4-D and 0.5 g/L MES buffer. ‘MS basal salt mixture’ was purchased from Sigma (No. M6899, St. Louis, MO, USA). Before autoclaving (121°C, 20 min), the pH was adjusted to 5.8 with 1 M KOH in both media. Both cultures were cultivated at 26°C in the dark on a rotary shaker (Infors, Bottmingen, Switzerland) operating at 180 rpm with a shaking diameter of 50 mm. The tobacco cells were sub-cultured weekly into fresh MS-medium by diluting 2.5 ml cell suspension into 47.5 ml fresh media. Modified MS-medium In a first experiment performed in the respiration activity monitoring system (RAMOS) device (39), plant cell growth in the commercial MSmedium was compared with plant cell growth in the MS-medium, which was produced from self-made stock solutions. The aim of this experiment was, to check if the plant cell growth is equal for both media. Both determined oxygen transfer rates did not deviate by more than 5% (data not shown); thus, it was proven that the commercial MS-medium can be reproduced from stock solutions. In order to modify the MS-medium different stock solutions were prepared which enabled a variation of single compounds. Different stock solutions were stored at 4°C. In the resulting modified MS-medium, the initial concentration of ammonium was increased from 20.6 mM to 30 mM and the initial potassium concentration was simultaneously decreased from 20.05 mM to 10.05 mM. Before autoclaving (121°C, 20 min), the pH was adjusted to 5.8 with 1 M KOH. Online measurement of the oxygen transfer rate (OTR) All experiments were conducted in a RAMOS device. The RAMOS device for the online measurement of the OTR in shake flasks was already introduced by Anderlei and Büchs (39) and Anderlei et al. (40). Measuring OTR online during cultivation is the most suitable way to quantify the physiological state of aerobic microorganisms and cell cultures since all metabolic steps are connected to oxygen. The benefits of the RAMOS device cultivating plant suspension cultures have been shown already in a previous project (41). BY-2 cells were cultivated in parallel in modified 250 ml RAMOS shake flasks. The shaker was operated in the dark at 180 rpm, 50 mm shaking diameter and at 26°C and all flasks had a filling volume of 50 ml. Since offline samples were needed, conventional nonmonitored Erlenmeyer shake flasks were used in addition to RAMOS flasks, inoculated by the same BY-2 cell master mix. In RAMOS flasks the hydrodynamic conditions and the concentrations in the gas-phase of the head space are the same as in regular Erlenmeyer flasks with cotton plugs (40), so that a same growth of the cell culture is assured. One flask was used for each sample in order to avoid a modification of the culture conditions due to the removed sample volume. Determination of different sugars BY-2 cell growth was monitored by determining fresh and dry weight. Fresh weight was analyzed by vacuum-filtrating 10 ml of cell suspension using a pre-weighed Whatman filter (No. 3, 55 mm diameter). The resulting biomass pellet was weighed (wet weight) and stored at 105°C in an oven until the mass, weighed with an electronic precision balance (SBC 31, Scaltec, Göttingen, Germany), remained constant (dry weight). Determination of different sugars Carbon source analysis was performed via high-performance liquid chromatography (HPLC). For the separation of the sample a carbohydrate column Pb2+ (No. 52898230, CS-Chromatography, Langerwehe, Germany) was used. The detection was carried out by a RI-Detector (Shodex, Tokyo, Japan), and for the sample analysis, the software Chromelion (Dionex, Idstein, Germany) was used. Determination of the osmolality The osmolality, measured via cryoscopy, was detected using Osmomat 030 (Gonotec, Berlin, Germany). After a 2-point calibration, the supernatant was analyzed according to the manufacturers' protocol. Determination of the conductivity and pH-value Both the conductivity and pHvalue were measured directly in the BY-2 cell culture broth. The conductivity was determined using the digital conductivity meter LF 340-A (WTW, Weilheim, Germany), and the pH-value was measured using pH-Meter pH510 (Eutech Instruments, Simi Valley, CA, USA). Photometric measurement of phosphate, ammonium and nitrate The phosphate concentration of the supernatant was measured using a round cuvette test kit (No. 1.00616.0001, Merck, Darmstadt, Germany) through detection of phosphomolybdenum blue (PMB) that is formed in the presence of phosphate ions, analogous to DIN EN 1189 D11. The ammonium concentration was quantified using a different round cuvette test (No. 1.14559.0001, Merck). Here, a blue indophenol derivate was detected analogous to DIN 38406 E5. The concentration of nitrate ions (No. 1.14883.0001, Merck), based on DIN 38405 D9, was determined by measuring the 4-nitro-2.6-dimethylphenol. All three ion concentrations were measured using Nova Spectroquant 60 (Merck).
FIG. 1. Nitrogen assimilation in plant cells.
Extraction of total soluble proteins BY-2 cells were filtrated out of the medium, and 0.1 g cells were resuspended in 0.6 ml extraction buffer (PBS pH 7.4 with 5 mM ethylenediaminetetraacetic acid (EDTA) and 5 mM ß-mercaptoethanol) and stored at − 20°C. The cells were thawed and destroyed (60 s, 55 W, duty cycle 0.7) via ultrasonication on ice (Labsonic U, B. Braun, Melsungen, Germany). Cell debris was removed by centrifugation (10 min, 14,000 rpm, 4°C) and the supernatant, containing the soluble proteins, was used for further analysis. Quantification of total protein amount The protein concentration was quantified using the Bradford assay (42). The respective concentrations were
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measured according to the manufacturer's microtiter plate protocol by using bovine serum albumin (BSA) as standard (Bio-Rad Laboratories, München, Germany). Data were evaluated applying the software Origin 8.5 by determining the slope by linear regression. SDS-PAGE and Western blot analysis Discontinuous SDS-polyacrylamide gels (43) were used for separating protein samples. Two micrograms protein were incubated with 2 μl of five-fold concentrated loading buffer (1 M Tris–HCl, SDS 4% (w/v), bromphenol blue 0.05% (w/v), β-mercaptoethanol 10% (w/v) and glycerol 30% (w/v)) in a total volume of 10 μl at 99°C for 10 min. NuPage® 4–12% (w/v) Bis–Tris gels (Invitrogen, Carlsbad, CA, USA) were prepared according to the manufacturer's protocol, and the prestained Fermentas PageRuler® marker (Fermentas, Glen Burnie, MD, USA) was used as a molecular weight standard. Purified TurboGFP (Evrogen, Moscow, Russia) and the supernatant of the wild-type BY-2 strain were used as positive and negative controls, respectively. For Western blot analysis, gels were transferred (30 V constant, 1 h) onto a nitrocellulose membrane (Whatman, Springfield Mill, UK). The membranes were blocked at room temperature with 5% (w/v) skim milk dissolved in PBS containing 0.05% (w/v) Tween-20 (PBST) for 30 min. Moreover, the membranes were incubated at 4°C over night with a rabbit polyclonal antibody against TurboGFP (Evrogen, Moscow, Russia) diluted 1:20,000 in PBS to detect TurboGFP. After the membrane was washed thrice with PBST, the membranes were then incubated with alkaline phosphatase (AP)-conjugated goat anti-rabbit immunoglobulin G (IgG) (Jackson ImmunoResearch, Suffolk, UK) diluted 1:5000 in PBS at room temperature for 1 h. Finally, bound antibodies were visualized by incubating the membrane for 10 min with NBT-BCIP diluted 1:100 in AP-buffer (100 mM Tris–HCl, 100 mM NaCl, 5 mM MgCl2, pH 9.6). The Western blot was analyzed densitometrically using the scanner ‘Perfection V700’ (Epson, Suwa, Japan). Band intensities were quantified using the software TotalLab TL100 (Nonlinear Dynamics, Newcastle, UK). Enzyme linked immunosorbent assay (ELISA) F-96 MaxiSorp microtiter plates (Nunc, Wiesbaden, Germany) were first coated with chicken anti hemagglutinin (HA) antibodies diluted in PBS buffer over night at 4°C. Free-binding sites were subsequently blocked with 3% (w/v) BSA dissolved in PBS. All following incubation steps were performed at room temperature with shaking. A serial dilution of HAstandard from 100 ng/ml to 1.5625 ng/ml as well as a serial dilution from the samples was applied to the wells. Then, the plates were incubated for 60 min. Afterwards those plates were incubated with murine anti-HA antibodies 9E7-37-37 (Benchmark Biolabs, Lincoln, NE, USA) diluted 1:2,000 in PBST for 60 min, followed by an incubation with goat anti-mouse (No. 31437, ThermoScientific, Rockford, IL, USA) polyclonal antibody diluted 1:5,000 in PBST for 60 min for the detection of the bound primary antibody. Bound secondary antibodies were detected by incubation with the ELISA substrate (Pierce No. 34028, ThermoScientific) for 30 min and finally the reaction was stopped using 1 N sulphuric acid. Between the different incubations steps, plates were washed thrice with PBST. Subsequently the absorbance at 405 nm was determined using the Synergy 4 microtiter plate reader (BioTek Instruments, VT, USA) and the binding data was evaluated using Origin 8.5 by determining the slope in the linear range of the dose– response curve by linear regression.
RESULTS AND DISCUSSION BY-2 cell growth characterization and product formation To study, in particular, BY-2 cell growth and product formation, BY-2 cells were cultivated in shake flasks at 26°C for 7 days. Fig. 2A represents the OTR, the concentration of phosphate ions and the concentration of the two nitrogen sources, ammonium and nitrate in the supernatant as a function of time (h). The OTR-curve is characterized by a monotonous increase. After 88 h (indicated by the dotted line) there is an interruption of the breathing activity by a reproducible characteristic small peak and a shift to a different breathing activity. After 103 h the OTR reached a maximum which corresponded with the depletion of the carbon sources (Fig. 2B). In biological systems, phosphate plays an important role in energy transfer via the formation of the pyrophosphate bond in ATP. The initial phosphate concentration in the MS-medium is 2.7 mM, and after approximately 74 h no more phosphate was detected in the supernatant. This depletion of phosphate was considered to be bound to myo-inositol to form phytic acid, which is the principal storage form of phosphorous in plants (44). Since the phosphate was bound there, it is no longer detectable with the used test kit. The two nitrogen sources in the MS-medium are ammonium (20.6 mM) and nitrate (39.4 mM). As seen in Fig. 2A, no more ammonium was detectable in the supernatant after 88 h. It can be seen that the depletion of ammonium correlated well with the shift in the OTR. In the same period the nitrate concentration decreased from its initial value to 24 mM. After ammonium was consumed by the cells
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FIG. 2. Cultivation of transgenic GFP-producing Nicotiana tabacum L. cv. BY-2 cells in MS-medium, (A) ammonium (diamonds), nitrate (squares) and phosphate (circles) concentration, oxygen transfer rate (OTR) (solid line), (B) total sugar concentration, OTR (solid line), (C) osmolality (down triangles), conductivity (circled dots), OTR (solid line), pH value (up triangles) and (D) dry weight (crosses) and OTR (solid line). Experimental conditions: flask volume, 250 ml; filling volume, 50 ml; initial sucrose concentration, 30 g/l; temperature, 26°C; shaking frequency, 180 rpm; shaking diameter, 5 cm. Dotted line indicates a reproducible change in metabolism.
the nitrate concentration dropped quickly and was depleted after approximately 122 h. Fig. 2B distinctly shows the sucrose depletion resulting from the hydrolysis of sucrose to its monomers glucose and fructose followed by their sequential consumption, with glucose being consumed first. The hydrolysis of sucrose is catalyzed by extracellular invertases (45) or by invertases located in the cell wall (46). Sucrose was entirely hydrolyzed after 95 h and glucose was consumed after 99 h so that the reproducible shift in the OTR can clearly be attributed to the depletion of ammonium. This experiment illustrated that once the carbon source was depleted, the OTR started decreasing. For tobacco suspension cultures in shake flasks 30 g/l sucrose is favorable, because the highest cell concentrations could be attained with this carbon source concentration (22). Using 30 g/l glucose instead of 30 g/l sucrose resulted in a slower cell growth (data not shown). The values for the osmolality and conductivity remained nearly constant in the first 72 h of the cultivation. Since the two monomers resulting from sucrose hydrolysis have a higher osmotic pressure than non-hydrolyzed sucrose, the osmolality remained nearly constant although carbon was consumed. After 72 h both values started decreasing and ended at values almost to zero at the end of the cultivation (Fig. 2C).
VOL. 113, 2012 This indicated that after 168 h roughly all nutrients in the medium were depleted and further cell growth and protein production was no longer possible. The culture broth was similar to “distilled water”. Since sugar is responsible for half of the osmolality, the decrease in the osmolality followed the decrease of the carbon source. After 120 h, no more phosphate, ammonium and nearly no nitrate was detectable in the medium (Fig. 2A). This correlated with low conductivity values at the end of the cultivation. Conductivity has also been used to monitor cell growth (47). A linear relationship between cell growth of N. tabacum cells and a decrease in conductivity was observed. One disadvantage of this approach is that this method is indirect and therefore the decrease in the dry weight at the end of batch fermentations was not monitored. This results maybe from the fact that cell lysis at the end of a fermentation is not monitored by conductivity measurements (48) (Fig. 2D). During batch cultivation, the culture showed a typical pH trend for BY-2 suspension cultures (Fig. 2C) (49). The pH dropped down from 5.7 to 4.5 during the first 48 h. Once ammonium was consumed by the cells (after 88 h) the pH-value increased from 4.5 to 5.5 at the end of the cultivation. The pH of the medium highly influences the availability of many minerals (50) since it changes the dissociation of the molecules. Hence, the uptake of nitrate and ammonium is markedly affected by the pH-value. Uptake of nitrate by BY-2 cells leads to a more alkaline pH of the medium, whereas ammonium uptake results in an acidification (51,52). The findings presented here clearly proved the acidification of the medium due to the ammonium consumption followed by the basification of the medium due to the total ammonium consumption which triggered the switch to nitrate as sole nitrogen source. This pH trend has also been shown for Ipomoea sp. cultures (53). Fig. 2D shows the OTR and the biomass concentration. The maximum of cell mass corresponded approximately with the highest value in the OTR and the depletion of the carbon source. A maximum value of 16.5 g/L cell dry weight was detected. In the last 48 h of the cultivation the dry weight decreased to a final concentration of 14 g/L. Once the carbon source was depleted, no more cell growth was observed. Influence of the nitrate to ammonium ratio on BY-2 cell growth Based on the previously discussed results, it was proven that the ammonium depletion and not a depletion of a carbon source led to the shift in the breathing activity, which is indicated in the OTR-curve by the dotted line (Fig. 2A). The correct balance of nitrate to ammonium ions is very important and has already been discussed in literature (24,25). Since the depletion of ammonium causes a reproducible change in the breathing activity the following experiment addresses how, in particular, the initial nitrate to ammonium ratio affects plant cell metabolism. Fig. 3 represents the oxygen transfer rates of transgenic BY2 cells in modified MS-medium containing decreasing initial concentrations of ammonium and simultaneously increasing concentrations of nitrate as a function of time. The reference culture (black line) contained 20.61 mM ammonium and 39.4 mM nitrate. A shift from ammonium to nitrate at a constant nitrogen concentration of 60 mM resulted in a lower growth rate. The growth rate as well as the highest OTR-value decreased with a decreased initial ammonium concentration. It can clearly be seen that supplying a mixture of both nitrate and ammonium resulted in better vegetative growth and enhanced nutrient assimilation than nitrate alone. These findings concur well with those of other studies (54,55). However, BY-2 cell can grow in a medium with ammonium as the sole nitrogen source if Krebs cycle acids such as citrate, malate, fumarate or succinate are added to the MS-medium (30). In the modified MS-medium containing 50 mM nitrate (gray) or containing nitrate as the sole nitrogen source (light gray) a significantly slower growth compared to that of the reference culture was observed. Supplying more ammonium gives the cell an advantage since the energy-consuming reduction of nitrate is avoided (Fig. 1). Based on these results no experiments were performed where the initial nitrate concentration was increased. It has clearly been shown that a higher initial ammonium concentration had a beneficial effect on plant cell growth. Moreover it is
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FIG. 3. Cultivation of transgenic Nicotiana tabacum BY-2 cells in modified MS-medium with decreasing initial ammonium concentrations. Oxygen transfer rates (OTR) of BY-2 cells growing in standard MS-medium containing 20.6 mM NH4 and 39.4 mM NO3 (black) and in modified MS-medium containing 10 mM NH4 and 50 mM KNO3 (gray line) or 60 mM KNO3 (light gray). Experimental conditions: Flask volume, 250 ml; filling volume, 50 ml; temperature, 26°C; shaking frequency; 180 rpm; shaking diameter, 5 cm.
known, that ammonium plays a key role in protein synthesis (30). Based on the previously discussed results it was worthwhile to investigate if an increase in the initial amount of ammonium has a beneficial effect on plant cell growth and protein production. This hypothesis has been investigated in the following experiments. Experiments with an increased initial ammonium concentration The impact of increasing the initial amount of ammonium in the MS-medium was investigated in a set of two experiments comparing the OTR in shake flasks using standard MS-medium with a modified MSmedium, inoculated with GFP-producing BY-2 cells. In the first experiment, the initial amount of ammonium was increased by adding of 20 mM ammonium chloride to the MS-medium; in a second experiment, the MS-medium was modified. Fig. 4 represents two OTR-curves obtained accordingly with the standard MS-medium and the ammonium-enriched medium. BY-2 cells cultivated in the ammonium-enriched medium showed a longer lag phase compared to cells cultivated in the standard MS-medium. The calculated osmolality in the ammonium-enriched MS-medium was 40 mOsm higher compared to that of the standard MS-medium. Since plant cells are highly osmosensitive, induced osmotic stress delayed BY-2 cell growth (56). Due to the prolonged lag phase, this type of modification was not a favorable option. Considering this
FIG. 4. Cultivation of transgenic GFP-producing Nicotiana tabacum L. cv. BY-2 cells in MS-medium (closed hexagons) and MS-medium with increased initial ammonium chloride concentration (open hexagons). Experimental conditions: flask volume, 250 ml; filling volume, 50 ml; initial sucrose concentration, 30 g/l; temperature, 26°C; shaking frequency, 180 rpm; shaking diameter, 5 cm.
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disadvantage, the intention was to increase the initial amount of ammonium in the MS-medium without increasing the initial osmotic pressure. To achieve this, ammonium was increased about 10 mM and potassium was decreased about 10 mM. In the modified MS-medium the initial amount of ammonium was 10 mM higher than that of standard MS-medium. Previous experiments with the BY-2 wild-type cell line showed that still 18.3 mM potassium was measured in the supernatant after 120 h of cultivation (data not shown). Fig. 5 depicts the data obtained with the standard MS-medium (solid symbols) and with the modified MS-medium (open symbols), where the osmotic pressure was kept constant by reducing the potassium concentration. Fig. 5A shows the impact of the higher initial amount of ammonium on the OTR. Despite the same initial biomass concentration, a different growth behavior was observed (Fig. 5A)
FIG. 5. Cultivation of transgenic GFP-producing Nicotiana tabacum BY-2 cells in MSmedium (closed symbols) and modified MS-medium (open symbols), (A) OTR (hexagons) and ammonium concentration (diamonds), (B) wet (down triangles) and dry weight (circles), (C) GFP (squares) and protein concentration (up triangles) related to cell broth. Experimental conditions: flask volume, 250 ml; filling volume, 50 ml; initial sucrose concentration, 30 g/l; temperature, 26°C; shaking frequency, 180 rpm; shaking diameter, 5 cm. Dotted lines correspond to the expected exhaustion of the ammonium concentration indicated by the bend in the OTR-curve.
J. BIOSCI. BIOENG., compared to the conventional MS-medium. It can be seen that the growth in the modified MS-medium was faster (μmax = 0.036 1/h in the modified MS-medium and 0.034 1/h in the standard MS-medium) and the OTRmax was higher. The characteristic shift in metabolism was shifted to a later time. It correlated with the exhaustion of the ammonium ions in the respective MS-medium. Reducing the initial amount of potassium to half had no detectable influence on BY-2 cell growth. Fig. 1 shows that ammonium as a readily metabolizable source of nitrogen can be incorporated directly into the nitrogen pathway. An increase of the initial nitrate concentration resulted in a significantly slower cell growth (Fig. 3). Thus, it is more advantageous for the BY-2 cells from an energy standpoint to use ammonium instead of nitrate because the energy consuming reducing step is avoided. Consuming ammonium directly resulted in faster growth and a higher biomass concentration (Fig. 5B). The total protein concentrations as well as the GFP concentration were significantly higher in the ammonium-enriched MS-medium compared to those in the standard MS-medium (Fig. 5C). After 144 h, the maximum GFP concentration nearly doubled (1.9-fold increase). Since ammonium can be used directly as nitrogen-source for the protein production, an increase in the initial amount of ammonium led to higher protein concentrations (27). Consequently, the ammonium increase in the modified MS-medium enhanced GFP production, too. Further offline parameters (conductivity, osmolality, pH-value, carbon source) have been also analyzed in the standard MS-medium as well as in the ammonium-enriched MS-medium. Between both media, no significant variations of the respective values have been observed (data not shown). In a final experiment it was investigated if this obtained knowledge can be transferred to another transgenic tobacco cell line to boost the production of a different recombinant protein. For this purpose the transgenic N. tabacum cell line CHA-13 was used, which was derived from the base cell culture NT-1. This base culture is again closely related to the BY-2 cell line. CHA-13 produces the pharmaceutically relevant protein influenza hemagglutinin (HA), which is targeted to endomembrane system of the plant cell. Fig. 6 represents the data obtained with the standard MS-medium (solid symbols) and with the modified (ammonium-enriched) MSmedium (open symbols) for the N. tabacum NT-1 cell line. The experimental set-up was the same as in the experiment, illustrated in Fig. 5. Fig. 6A shows the OTR-curves and the ammonium concentration of the NT-1 cell culture supernatant in the standard and the ammonium-enriched MS-medium. In the first 82 h of the cultivation, the growth pattern in both MS-media was identical. Once ammonium was totally consumed in the standard MS-medium the OTR of the NT-1 cells declined. As a result the biomass concentration of the cells cultivated in the standard MS-medium was lower (Fig. 6B). Fig. 6C depicts the total protein concentration together with the concentration of the influenza hemagglutinin (HA) protein. As previously demonstrated for the BY-2 cell line (Fig. 5C), the positive effect of the ammonium-enriched medium on the protein and target protein formation was also confirmed for the NT-1 cell line. After 120 h the HA concentration also doubled. During the last 48 h the HA concentration started declining which could be the result of a proteolytic degradation of the protein (57,58). The GFP concentration, however, remained rather constant until the end of the cultivation which could be explained by the remarkably stable structure of GFP (59). The trend of the protein concentration was not completely reproducible. The reason for that is not yet fully understood and will be the subject of further experiments. In summary, this investigation has shown for the first time a comprehensive characterization of the transgenic Nicotiana tabacum BY-2 cell growth by combining online and offline analysis of multiple parameters. Furthermore, the MS-medium has been improved by the addition of ammonium which resulted in a two fold increase of the target protein GFP. The RAMOS device, which allows the online analysis
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and acknowledge the company Dow AgroSciences LLC for the funding of this research project. References
FIG. 6. Cultivation of transgenic HA-producing Nicotiana tabacum NT-1 cells in MSmedium (closed symbols) and modified MS-medium (open symbols), (A) OTR (hexagons) and ammonium concentration (diamonds), (B) wet (down triangles) and dry weight (circles), (C) HA (squares) and protein concentration (up triangles) related to cell broth. Experimental conditions: flask volume, 250 ml; filling volume, 50 ml; initial sucrose concentration, 30 g/l; temperature, 26°C; shaking frequency; 180 rpm; shaking diameter, 5 cm. Dotted lines represent the expected exhaustion of the ammonium concentration indicated by the bend in the OTR-curve. Due to an electrical power breakdown there was no data logging of the OTR between 41 h and 51 h.
of oxygen consumption has been proven to be a useful analytic tool, since changes of cell metabolism could be easily detected online. For the first time an online detection of the ammonium depletion based on the OTR is reported for a cultivation of N. tabacum BY-2 cells. A further analysis of the nutrients could show the depletion of ammonia as reason for that metabolic shift. Furthermore, this improved medium was successfully used for another transgenic tobacco cell line to characterize the growth behavior and to boost target product formation of the pharmaceutically relevant protein influenza hemagglutinin (HA). ACKNOWLEDGMENTS The authors gratefully thank Dr. Flora Schuster (RWTH Aachen University) for her excellent assistance with the plant tissue culture
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Comprehensive characterization of two different Nicotiana tabacum cell lines leads to doubled GFP and HA protein production by media optimization David A. Ullisch, 1 Christina A. Müller, 1 Sabrina Maibaum, 1 Janina Kirchhoff, 2 Andreas Schiermeyer, 2 Stefan Schillberg, 2 Jean L. Roberts, 3 Wiltrud Treffenfeldt, 3 and Jochen Büchs 1,⁎ RWTH Aachen University, AVT – Biochemical Engineering, Worringer Weg 1, 52056 Aachen, Germany, 1 Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Department Plant Biotechnology, Forckenbeckstrasse 6, 52074 Aachen, Germany, 2 and Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA 3 Received 23 May 2011; accepted 29 September 2011 Available online 4 November 2011
For over two decades, plant cell cultures have been a promising research platform to express recombinant and therapeutic proteins such as hormones, growth factors, full-size antibodies and antigens. Chosen as a good host for manufacturing recombinant proteins, the Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cell line has been studied in shake flasks by offline analysis of only a few growth parameters. The objective of this study is to comprehensively characterize the growth of a transgenic BY-2 cell line and to investigate the expression profile of the model protein GFP. Based on the correlations between nutrient consumption, cell growth and product formation, the intention is to improve the standard MS-medium. Hereby, multiple growth parameters were analyzed offline and online by using a respiration activity monitoring system (RAMOS). A reproducibly observed shift of the oxygen transfer rate (OTR) could be identified to indicate ammonium depletion in the medium. Concurrent with this ammonium depletion, the total protein concentration began to decrease. After the MS-medium was improved, the GFP concentration nearly doubled. When this improved ammonium enriched medium was applied to another transgenic tobacco cell line similar improvements to the amount of the glycoprotein influenza hemagglutinin (HA) produced by Nicotiana tabacum NT-1 cells could be achieved. Ultimately, this combined offline and online analysis can be successfully used for further cell line characterization and media optimization to improve growth and boost target product formation. © 2011, The Society for Biotechnology, Japan. All rights reserved. [Keywords: BY-2 cells; Influenza hemagglutinin; Media optimization; Nitrogen source; Oxygen transfer rate; Plant cell culture; respiration activity monitoring system (RAMOS); Shake flasks]
The number of recombinant proteins produced in plants has steadily increased since the first recombinant protein was expressed in a plant cell culture in the early nineties (1). After this breakthrough, different transgenic plant cell suspension cultures have been used producing recombinant proteins including rice (2), soybeans (3) and tomatoes (4). The production of recombinant proteins from transgenic suspension cultures offers many advantages over that from intact transgenic plants including a better process control and fast generation of transgenic cell lines (5). Another advantage is, that the target protein can be potentially secreted to the culture broth which facilitates the separation and purification. In addition plant cell cultures are not exposed to agrochemicals and variable cultivation conditions due to changes of weather and other environmental conditions (6) and moreover plant cells have their well-documented ability to perform post-translational modifications (7). The tobacco cell line Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2), first isolated by Kato et al. (8) , was chosen as a preferred host cell line for ⁎ Corresponding author. Tel.: + 49 241 8023569; fax: + 49 241 8022570. E-mail address: [email protected] (J. Büchs). Abbreviations: BY-2, Nicotiana tabacum L. cv. Bright Yellow 2; NT-1, Nicotiana tabacum NT-1; OTR, oxygen transfer rate; PBS, phosphate buffered saline; PBST, phosphate buffered saline containing 0.05% (w/v) Tween 20; RAMOS, respiration activity monitoring system.
plant suspension cultures, since this cell line is well characterized — showing high growth rates and high cell synchrony (9). According to Combettes et al. (10) and David and Perrot-Rechenmann (11) this cell line is the model cell line for studying of plant cell cycles and investigating the cellular biology of plants. Up to now, a number of various recombinant proteins, such as antibody fragments (12,13), fullsize antibodies (14,15), enzymes (16–18) and even mature immunologically active allergens (19) have been produced in BY-2 cells. Species-specific nutrient requirements have been described for a wide range of different plant cell cultures such as rice (20), soybeans (21) and tobacco (22). Therefore, different basal salt media have been empirically developed solely by trial and error, and subsequently dose–response experiments have been conducted. Nitrogen – which is the macronutrient with the highest concentration in media – is a constituent of both nucleic acids and proteins and is thus essential to plant life. Nitrogen plays a pivotal role in plant cell metabolism and is directly connected to amino acid and protein biosynthesis (23). Most commonly used nitrogen sources in media for cultivating plant cells are nitrate, ammonium, urea and amino acids. The MS-medium (22) consists of a mixture of two nitrogen sources in a defined ratio, namely nitrate and ammonium. The ratio of nitrate to ammonium ions plays an important role in the metabolism of different plants and has been already discussed in literature (24,25). The assimilation of
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ammonium and nitrate ions is shown in Fig. 1. Ammonium is a readily metabolizable source of nitrogen and is directly transformed into glutamine and glutamic acid by glutamine synthetase (GS) and glutamate synthase (GOGAT) (26). However, cells cannot grow in a medium with ammonium as the sole nitrogen source (27). Explanations for that are the latent toxicity induced by an excess of ammonium ions and the need to control the pH (28). Without control, the pH of the media containing ammonium as the sole nitrogen source falls rapidly to a point where no cell growth is possible (29). But if Krebs cycle acids such as citrate, malate, fumarate or succinate are added to the MSmedium plant cell growth can be observed (30). Nitrate, in contrast, has to be reduced to ammonium before being utilized (Fig. 1). Reduction of ammonium to nitrate is a two-step reaction catalyzed by the nitrate reductase (NR) and by the nitrite reductase (NiR). This reduction is performed by the cell at the expense of 4 mol of NAD(P)H per mol of nitrate (31). On the one hand the presence of nitrate induces both enzymes (32,33); on the other hand ammonium and amino acids can inhibit the activity of these enzymes (34,35). Thus, the pathway is regulated by its substrates and products. Some plants can grow in a medium with nitrate as sole nitrogen source, but BY-2 cell growth is better when the medium contains a mixture of nitrate and ammonium (30). Urea, however, can be used as a sole nitrogen source, but the growth is slower than that in a mixture of ammonium and nitrate (36). Up to now, individual studies have only handled one or two parameters (i.e., biomass or osmolality or conductivity) for studying cell growth. Such limited studies, however, do not provide a comprehensive insight into BY-2 cell metabolism. Moreover, even though the MS-medium has been optimized for rapid tobacco cell growth (22), it has not been optimized for recombinant protein formation. Thus, the objective of this study is to assay multiple growth parameters both offline and online in order to gain a deeper insight into BY-2 cell metabolism. A further objective is to optimize the standard MS-medium to increase target protein production. MATERIALS AND METHODS Plant cell lines Suspension cultures of the transgenic Nicotiana tabacum L. cv. BY-2 cell line expressing GFP were developed from wild-type strain N. tabacum L. cv. BY-2. This wild-type strain was transformed using Agrobacterium tumefaciens (strain LBA4404) containing the vector pDAB100352. A. tumefaciens harboring pDAB100352 was co-incubated with wild-type N. tabacum BY-2 suspension cultures. The transformed plant cells were selected using the herbicide imazethapyr. The T-DNA of pDAB100352 encodes the expression cassette for ER-retarded (endoplasmatic reticulum) green fluorescent protein (GFP-KDEL) (37) under the control of the Cassava vein mosaic virus (CsVMV) promoter (38). The transgenic N. tabacum NT-1 cell line expressing the glycoprotein influenza hemagglutinin (HA) contains the plasmid previously described (Cardineau, G. A., Manson, H. S., VanEck, J. M., Kirk, D. D., and Walmsley, A. M., US patent 2009/0017065 A1, 2009). The expression of HA was also driven
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by the Cassava vein mosaic virus promoter and the recombinant HA was targeted to the endomembrane system of the plant cell. General cultivation conditions BY-2 suspension cultures (250 ml Erlenmeyer flasks with a filling volume of 50 ml) were maintained in MS-medium (22), supplemented with 30 g/l sucrose, 0.2 g/l KH2PO4, 0.6 mg/l thiamine-HCl and 0.2 mg/l 2.4-D. NT-1 suspension cultures (250 ml Erlenmeyer flasks with a filling volume of 50 ml), however, were maintained in MS-medium, supplemented with 30 g/l sucrose, 274.8 mg/l K2HPO4, 170 mg/l KH2PO4, 1 mg/l thiamine-HCL, 4.44 mg/l 2.4-D and 0.5 g/L MES buffer. ‘MS basal salt mixture’ was purchased from Sigma (No. M6899, St. Louis, MO, USA). Before autoclaving (121°C, 20 min), the pH was adjusted to 5.8 with 1 M KOH in both media. Both cultures were cultivated at 26°C in the dark on a rotary shaker (Infors, Bottmingen, Switzerland) operating at 180 rpm with a shaking diameter of 50 mm. The tobacco cells were sub-cultured weekly into fresh MS-medium by diluting 2.5 ml cell suspension into 47.5 ml fresh media. Modified MS-medium In a first experiment performed in the respiration activity monitoring system (RAMOS) device (39), plant cell growth in the commercial MSmedium was compared with plant cell growth in the MS-medium, which was produced from self-made stock solutions. The aim of this experiment was, to check if the plant cell growth is equal for both media. Both determined oxygen transfer rates did not deviate by more than 5% (data not shown); thus, it was proven that the commercial MS-medium can be reproduced from stock solutions. In order to modify the MS-medium different stock solutions were prepared which enabled a variation of single compounds. Different stock solutions were stored at 4°C. In the resulting modified MS-medium, the initial concentration of ammonium was increased from 20.6 mM to 30 mM and the initial potassium concentration was simultaneously decreased from 20.05 mM to 10.05 mM. Before autoclaving (121°C, 20 min), the pH was adjusted to 5.8 with 1 M KOH. Online measurement of the oxygen transfer rate (OTR) All experiments were conducted in a RAMOS device. The RAMOS device for the online measurement of the OTR in shake flasks was already introduced by Anderlei and Büchs (39) and Anderlei et al. (40). Measuring OTR online during cultivation is the most suitable way to quantify the physiological state of aerobic microorganisms and cell cultures since all metabolic steps are connected to oxygen. The benefits of the RAMOS device cultivating plant suspension cultures have been shown already in a previous project (41). BY-2 cells were cultivated in parallel in modified 250 ml RAMOS shake flasks. The shaker was operated in the dark at 180 rpm, 50 mm shaking diameter and at 26°C and all flasks had a filling volume of 50 ml. Since offline samples were needed, conventional nonmonitored Erlenmeyer shake flasks were used in addition to RAMOS flasks, inoculated by the same BY-2 cell master mix. In RAMOS flasks the hydrodynamic conditions and the concentrations in the gas-phase of the head space are the same as in regular Erlenmeyer flasks with cotton plugs (40), so that a same growth of the cell culture is assured. One flask was used for each sample in order to avoid a modification of the culture conditions due to the removed sample volume. Determination of different sugars BY-2 cell growth was monitored by determining fresh and dry weight. Fresh weight was analyzed by vacuum-filtrating 10 ml of cell suspension using a pre-weighed Whatman filter (No. 3, 55 mm diameter). The resulting biomass pellet was weighed (wet weight) and stored at 105°C in an oven until the mass, weighed with an electronic precision balance (SBC 31, Scaltec, Göttingen, Germany), remained constant (dry weight). Determination of different sugars Carbon source analysis was performed via high-performance liquid chromatography (HPLC). For the separation of the sample a carbohydrate column Pb2+ (No. 52898230, CS-Chromatography, Langerwehe, Germany) was used. The detection was carried out by a RI-Detector (Shodex, Tokyo, Japan), and for the sample analysis, the software Chromelion (Dionex, Idstein, Germany) was used. Determination of the osmolality The osmolality, measured via cryoscopy, was detected using Osmomat 030 (Gonotec, Berlin, Germany). After a 2-point calibration, the supernatant was analyzed according to the manufacturers' protocol. Determination of the conductivity and pH-value Both the conductivity and pHvalue were measured directly in the BY-2 cell culture broth. The conductivity was determined using the digital conductivity meter LF 340-A (WTW, Weilheim, Germany), and the pH-value was measured using pH-Meter pH510 (Eutech Instruments, Simi Valley, CA, USA). Photometric measurement of phosphate, ammonium and nitrate The phosphate concentration of the supernatant was measured using a round cuvette test kit (No. 1.00616.0001, Merck, Darmstadt, Germany) through detection of phosphomolybdenum blue (PMB) that is formed in the presence of phosphate ions, analogous to DIN EN 1189 D11. The ammonium concentration was quantified using a different round cuvette test (No. 1.14559.0001, Merck). Here, a blue indophenol derivate was detected analogous to DIN 38406 E5. The concentration of nitrate ions (No. 1.14883.0001, Merck), based on DIN 38405 D9, was determined by measuring the 4-nitro-2.6-dimethylphenol. All three ion concentrations were measured using Nova Spectroquant 60 (Merck).
FIG. 1. Nitrogen assimilation in plant cells.
Extraction of total soluble proteins BY-2 cells were filtrated out of the medium, and 0.1 g cells were resuspended in 0.6 ml extraction buffer (PBS pH 7.4 with 5 mM ethylenediaminetetraacetic acid (EDTA) and 5 mM ß-mercaptoethanol) and stored at − 20°C. The cells were thawed and destroyed (60 s, 55 W, duty cycle 0.7) via ultrasonication on ice (Labsonic U, B. Braun, Melsungen, Germany). Cell debris was removed by centrifugation (10 min, 14,000 rpm, 4°C) and the supernatant, containing the soluble proteins, was used for further analysis. Quantification of total protein amount The protein concentration was quantified using the Bradford assay (42). The respective concentrations were
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measured according to the manufacturer's microtiter plate protocol by using bovine serum albumin (BSA) as standard (Bio-Rad Laboratories, München, Germany). Data were evaluated applying the software Origin 8.5 by determining the slope by linear regression. SDS-PAGE and Western blot analysis Discontinuous SDS-polyacrylamide gels (43) were used for separating protein samples. Two micrograms protein were incubated with 2 μl of five-fold concentrated loading buffer (1 M Tris–HCl, SDS 4% (w/v), bromphenol blue 0.05% (w/v), β-mercaptoethanol 10% (w/v) and glycerol 30% (w/v)) in a total volume of 10 μl at 99°C for 10 min. NuPage® 4–12% (w/v) Bis–Tris gels (Invitrogen, Carlsbad, CA, USA) were prepared according to the manufacturer's protocol, and the prestained Fermentas PageRuler® marker (Fermentas, Glen Burnie, MD, USA) was used as a molecular weight standard. Purified TurboGFP (Evrogen, Moscow, Russia) and the supernatant of the wild-type BY-2 strain were used as positive and negative controls, respectively. For Western blot analysis, gels were transferred (30 V constant, 1 h) onto a nitrocellulose membrane (Whatman, Springfield Mill, UK). The membranes were blocked at room temperature with 5% (w/v) skim milk dissolved in PBS containing 0.05% (w/v) Tween-20 (PBST) for 30 min. Moreover, the membranes were incubated at 4°C over night with a rabbit polyclonal antibody against TurboGFP (Evrogen, Moscow, Russia) diluted 1:20,000 in PBS to detect TurboGFP. After the membrane was washed thrice with PBST, the membranes were then incubated with alkaline phosphatase (AP)-conjugated goat anti-rabbit immunoglobulin G (IgG) (Jackson ImmunoResearch, Suffolk, UK) diluted 1:5000 in PBS at room temperature for 1 h. Finally, bound antibodies were visualized by incubating the membrane for 10 min with NBT-BCIP diluted 1:100 in AP-buffer (100 mM Tris–HCl, 100 mM NaCl, 5 mM MgCl2, pH 9.6). The Western blot was analyzed densitometrically using the scanner ‘Perfection V700’ (Epson, Suwa, Japan). Band intensities were quantified using the software TotalLab TL100 (Nonlinear Dynamics, Newcastle, UK). Enzyme linked immunosorbent assay (ELISA) F-96 MaxiSorp microtiter plates (Nunc, Wiesbaden, Germany) were first coated with chicken anti hemagglutinin (HA) antibodies diluted in PBS buffer over night at 4°C. Free-binding sites were subsequently blocked with 3% (w/v) BSA dissolved in PBS. All following incubation steps were performed at room temperature with shaking. A serial dilution of HAstandard from 100 ng/ml to 1.5625 ng/ml as well as a serial dilution from the samples was applied to the wells. Then, the plates were incubated for 60 min. Afterwards those plates were incubated with murine anti-HA antibodies 9E7-37-37 (Benchmark Biolabs, Lincoln, NE, USA) diluted 1:2,000 in PBST for 60 min, followed by an incubation with goat anti-mouse (No. 31437, ThermoScientific, Rockford, IL, USA) polyclonal antibody diluted 1:5,000 in PBST for 60 min for the detection of the bound primary antibody. Bound secondary antibodies were detected by incubation with the ELISA substrate (Pierce No. 34028, ThermoScientific) for 30 min and finally the reaction was stopped using 1 N sulphuric acid. Between the different incubations steps, plates were washed thrice with PBST. Subsequently the absorbance at 405 nm was determined using the Synergy 4 microtiter plate reader (BioTek Instruments, VT, USA) and the binding data was evaluated using Origin 8.5 by determining the slope in the linear range of the dose– response curve by linear regression.
RESULTS AND DISCUSSION BY-2 cell growth characterization and product formation To study, in particular, BY-2 cell growth and product formation, BY-2 cells were cultivated in shake flasks at 26°C for 7 days. Fig. 2A represents the OTR, the concentration of phosphate ions and the concentration of the two nitrogen sources, ammonium and nitrate in the supernatant as a function of time (h). The OTR-curve is characterized by a monotonous increase. After 88 h (indicated by the dotted line) there is an interruption of the breathing activity by a reproducible characteristic small peak and a shift to a different breathing activity. After 103 h the OTR reached a maximum which corresponded with the depletion of the carbon sources (Fig. 2B). In biological systems, phosphate plays an important role in energy transfer via the formation of the pyrophosphate bond in ATP. The initial phosphate concentration in the MS-medium is 2.7 mM, and after approximately 74 h no more phosphate was detected in the supernatant. This depletion of phosphate was considered to be bound to myo-inositol to form phytic acid, which is the principal storage form of phosphorous in plants (44). Since the phosphate was bound there, it is no longer detectable with the used test kit. The two nitrogen sources in the MS-medium are ammonium (20.6 mM) and nitrate (39.4 mM). As seen in Fig. 2A, no more ammonium was detectable in the supernatant after 88 h. It can be seen that the depletion of ammonium correlated well with the shift in the OTR. In the same period the nitrate concentration decreased from its initial value to 24 mM. After ammonium was consumed by the cells
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FIG. 2. Cultivation of transgenic GFP-producing Nicotiana tabacum L. cv. BY-2 cells in MS-medium, (A) ammonium (diamonds), nitrate (squares) and phosphate (circles) concentration, oxygen transfer rate (OTR) (solid line), (B) total sugar concentration, OTR (solid line), (C) osmolality (down triangles), conductivity (circled dots), OTR (solid line), pH value (up triangles) and (D) dry weight (crosses) and OTR (solid line). Experimental conditions: flask volume, 250 ml; filling volume, 50 ml; initial sucrose concentration, 30 g/l; temperature, 26°C; shaking frequency, 180 rpm; shaking diameter, 5 cm. Dotted line indicates a reproducible change in metabolism.
the nitrate concentration dropped quickly and was depleted after approximately 122 h. Fig. 2B distinctly shows the sucrose depletion resulting from the hydrolysis of sucrose to its monomers glucose and fructose followed by their sequential consumption, with glucose being consumed first. The hydrolysis of sucrose is catalyzed by extracellular invertases (45) or by invertases located in the cell wall (46). Sucrose was entirely hydrolyzed after 95 h and glucose was consumed after 99 h so that the reproducible shift in the OTR can clearly be attributed to the depletion of ammonium. This experiment illustrated that once the carbon source was depleted, the OTR started decreasing. For tobacco suspension cultures in shake flasks 30 g/l sucrose is favorable, because the highest cell concentrations could be attained with this carbon source concentration (22). Using 30 g/l glucose instead of 30 g/l sucrose resulted in a slower cell growth (data not shown). The values for the osmolality and conductivity remained nearly constant in the first 72 h of the cultivation. Since the two monomers resulting from sucrose hydrolysis have a higher osmotic pressure than non-hydrolyzed sucrose, the osmolality remained nearly constant although carbon was consumed. After 72 h both values started decreasing and ended at values almost to zero at the end of the cultivation (Fig. 2C).
VOL. 113, 2012 This indicated that after 168 h roughly all nutrients in the medium were depleted and further cell growth and protein production was no longer possible. The culture broth was similar to “distilled water”. Since sugar is responsible for half of the osmolality, the decrease in the osmolality followed the decrease of the carbon source. After 120 h, no more phosphate, ammonium and nearly no nitrate was detectable in the medium (Fig. 2A). This correlated with low conductivity values at the end of the cultivation. Conductivity has also been used to monitor cell growth (47). A linear relationship between cell growth of N. tabacum cells and a decrease in conductivity was observed. One disadvantage of this approach is that this method is indirect and therefore the decrease in the dry weight at the end of batch fermentations was not monitored. This results maybe from the fact that cell lysis at the end of a fermentation is not monitored by conductivity measurements (48) (Fig. 2D). During batch cultivation, the culture showed a typical pH trend for BY-2 suspension cultures (Fig. 2C) (49). The pH dropped down from 5.7 to 4.5 during the first 48 h. Once ammonium was consumed by the cells (after 88 h) the pH-value increased from 4.5 to 5.5 at the end of the cultivation. The pH of the medium highly influences the availability of many minerals (50) since it changes the dissociation of the molecules. Hence, the uptake of nitrate and ammonium is markedly affected by the pH-value. Uptake of nitrate by BY-2 cells leads to a more alkaline pH of the medium, whereas ammonium uptake results in an acidification (51,52). The findings presented here clearly proved the acidification of the medium due to the ammonium consumption followed by the basification of the medium due to the total ammonium consumption which triggered the switch to nitrate as sole nitrogen source. This pH trend has also been shown for Ipomoea sp. cultures (53). Fig. 2D shows the OTR and the biomass concentration. The maximum of cell mass corresponded approximately with the highest value in the OTR and the depletion of the carbon source. A maximum value of 16.5 g/L cell dry weight was detected. In the last 48 h of the cultivation the dry weight decreased to a final concentration of 14 g/L. Once the carbon source was depleted, no more cell growth was observed. Influence of the nitrate to ammonium ratio on BY-2 cell growth Based on the previously discussed results, it was proven that the ammonium depletion and not a depletion of a carbon source led to the shift in the breathing activity, which is indicated in the OTR-curve by the dotted line (Fig. 2A). The correct balance of nitrate to ammonium ions is very important and has already been discussed in literature (24,25). Since the depletion of ammonium causes a reproducible change in the breathing activity the following experiment addresses how, in particular, the initial nitrate to ammonium ratio affects plant cell metabolism. Fig. 3 represents the oxygen transfer rates of transgenic BY2 cells in modified MS-medium containing decreasing initial concentrations of ammonium and simultaneously increasing concentrations of nitrate as a function of time. The reference culture (black line) contained 20.61 mM ammonium and 39.4 mM nitrate. A shift from ammonium to nitrate at a constant nitrogen concentration of 60 mM resulted in a lower growth rate. The growth rate as well as the highest OTR-value decreased with a decreased initial ammonium concentration. It can clearly be seen that supplying a mixture of both nitrate and ammonium resulted in better vegetative growth and enhanced nutrient assimilation than nitrate alone. These findings concur well with those of other studies (54,55). However, BY-2 cell can grow in a medium with ammonium as the sole nitrogen source if Krebs cycle acids such as citrate, malate, fumarate or succinate are added to the MS-medium (30). In the modified MS-medium containing 50 mM nitrate (gray) or containing nitrate as the sole nitrogen source (light gray) a significantly slower growth compared to that of the reference culture was observed. Supplying more ammonium gives the cell an advantage since the energy-consuming reduction of nitrate is avoided (Fig. 1). Based on these results no experiments were performed where the initial nitrate concentration was increased. It has clearly been shown that a higher initial ammonium concentration had a beneficial effect on plant cell growth. Moreover it is
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FIG. 3. Cultivation of transgenic Nicotiana tabacum BY-2 cells in modified MS-medium with decreasing initial ammonium concentrations. Oxygen transfer rates (OTR) of BY-2 cells growing in standard MS-medium containing 20.6 mM NH4 and 39.4 mM NO3 (black) and in modified MS-medium containing 10 mM NH4 and 50 mM KNO3 (gray line) or 60 mM KNO3 (light gray). Experimental conditions: Flask volume, 250 ml; filling volume, 50 ml; temperature, 26°C; shaking frequency; 180 rpm; shaking diameter, 5 cm.
known, that ammonium plays a key role in protein synthesis (30). Based on the previously discussed results it was worthwhile to investigate if an increase in the initial amount of ammonium has a beneficial effect on plant cell growth and protein production. This hypothesis has been investigated in the following experiments. Experiments with an increased initial ammonium concentration The impact of increasing the initial amount of ammonium in the MS-medium was investigated in a set of two experiments comparing the OTR in shake flasks using standard MS-medium with a modified MSmedium, inoculated with GFP-producing BY-2 cells. In the first experiment, the initial amount of ammonium was increased by adding of 20 mM ammonium chloride to the MS-medium; in a second experiment, the MS-medium was modified. Fig. 4 represents two OTR-curves obtained accordingly with the standard MS-medium and the ammonium-enriched medium. BY-2 cells cultivated in the ammonium-enriched medium showed a longer lag phase compared to cells cultivated in the standard MS-medium. The calculated osmolality in the ammonium-enriched MS-medium was 40 mOsm higher compared to that of the standard MS-medium. Since plant cells are highly osmosensitive, induced osmotic stress delayed BY-2 cell growth (56). Due to the prolonged lag phase, this type of modification was not a favorable option. Considering this
FIG. 4. Cultivation of transgenic GFP-producing Nicotiana tabacum L. cv. BY-2 cells in MS-medium (closed hexagons) and MS-medium with increased initial ammonium chloride concentration (open hexagons). Experimental conditions: flask volume, 250 ml; filling volume, 50 ml; initial sucrose concentration, 30 g/l; temperature, 26°C; shaking frequency, 180 rpm; shaking diameter, 5 cm.
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disadvantage, the intention was to increase the initial amount of ammonium in the MS-medium without increasing the initial osmotic pressure. To achieve this, ammonium was increased about 10 mM and potassium was decreased about 10 mM. In the modified MS-medium the initial amount of ammonium was 10 mM higher than that of standard MS-medium. Previous experiments with the BY-2 wild-type cell line showed that still 18.3 mM potassium was measured in the supernatant after 120 h of cultivation (data not shown). Fig. 5 depicts the data obtained with the standard MS-medium (solid symbols) and with the modified MS-medium (open symbols), where the osmotic pressure was kept constant by reducing the potassium concentration. Fig. 5A shows the impact of the higher initial amount of ammonium on the OTR. Despite the same initial biomass concentration, a different growth behavior was observed (Fig. 5A)
FIG. 5. Cultivation of transgenic GFP-producing Nicotiana tabacum BY-2 cells in MSmedium (closed symbols) and modified MS-medium (open symbols), (A) OTR (hexagons) and ammonium concentration (diamonds), (B) wet (down triangles) and dry weight (circles), (C) GFP (squares) and protein concentration (up triangles) related to cell broth. Experimental conditions: flask volume, 250 ml; filling volume, 50 ml; initial sucrose concentration, 30 g/l; temperature, 26°C; shaking frequency, 180 rpm; shaking diameter, 5 cm. Dotted lines correspond to the expected exhaustion of the ammonium concentration indicated by the bend in the OTR-curve.
J. BIOSCI. BIOENG., compared to the conventional MS-medium. It can be seen that the growth in the modified MS-medium was faster (μmax = 0.036 1/h in the modified MS-medium and 0.034 1/h in the standard MS-medium) and the OTRmax was higher. The characteristic shift in metabolism was shifted to a later time. It correlated with the exhaustion of the ammonium ions in the respective MS-medium. Reducing the initial amount of potassium to half had no detectable influence on BY-2 cell growth. Fig. 1 shows that ammonium as a readily metabolizable source of nitrogen can be incorporated directly into the nitrogen pathway. An increase of the initial nitrate concentration resulted in a significantly slower cell growth (Fig. 3). Thus, it is more advantageous for the BY-2 cells from an energy standpoint to use ammonium instead of nitrate because the energy consuming reducing step is avoided. Consuming ammonium directly resulted in faster growth and a higher biomass concentration (Fig. 5B). The total protein concentrations as well as the GFP concentration were significantly higher in the ammonium-enriched MS-medium compared to those in the standard MS-medium (Fig. 5C). After 144 h, the maximum GFP concentration nearly doubled (1.9-fold increase). Since ammonium can be used directly as nitrogen-source for the protein production, an increase in the initial amount of ammonium led to higher protein concentrations (27). Consequently, the ammonium increase in the modified MS-medium enhanced GFP production, too. Further offline parameters (conductivity, osmolality, pH-value, carbon source) have been also analyzed in the standard MS-medium as well as in the ammonium-enriched MS-medium. Between both media, no significant variations of the respective values have been observed (data not shown). In a final experiment it was investigated if this obtained knowledge can be transferred to another transgenic tobacco cell line to boost the production of a different recombinant protein. For this purpose the transgenic N. tabacum cell line CHA-13 was used, which was derived from the base cell culture NT-1. This base culture is again closely related to the BY-2 cell line. CHA-13 produces the pharmaceutically relevant protein influenza hemagglutinin (HA), which is targeted to endomembrane system of the plant cell. Fig. 6 represents the data obtained with the standard MS-medium (solid symbols) and with the modified (ammonium-enriched) MSmedium (open symbols) for the N. tabacum NT-1 cell line. The experimental set-up was the same as in the experiment, illustrated in Fig. 5. Fig. 6A shows the OTR-curves and the ammonium concentration of the NT-1 cell culture supernatant in the standard and the ammonium-enriched MS-medium. In the first 82 h of the cultivation, the growth pattern in both MS-media was identical. Once ammonium was totally consumed in the standard MS-medium the OTR of the NT-1 cells declined. As a result the biomass concentration of the cells cultivated in the standard MS-medium was lower (Fig. 6B). Fig. 6C depicts the total protein concentration together with the concentration of the influenza hemagglutinin (HA) protein. As previously demonstrated for the BY-2 cell line (Fig. 5C), the positive effect of the ammonium-enriched medium on the protein and target protein formation was also confirmed for the NT-1 cell line. After 120 h the HA concentration also doubled. During the last 48 h the HA concentration started declining which could be the result of a proteolytic degradation of the protein (57,58). The GFP concentration, however, remained rather constant until the end of the cultivation which could be explained by the remarkably stable structure of GFP (59). The trend of the protein concentration was not completely reproducible. The reason for that is not yet fully understood and will be the subject of further experiments. In summary, this investigation has shown for the first time a comprehensive characterization of the transgenic Nicotiana tabacum BY-2 cell growth by combining online and offline analysis of multiple parameters. Furthermore, the MS-medium has been improved by the addition of ammonium which resulted in a two fold increase of the target protein GFP. The RAMOS device, which allows the online analysis
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and acknowledge the company Dow AgroSciences LLC for the funding of this research project. References
FIG. 6. Cultivation of transgenic HA-producing Nicotiana tabacum NT-1 cells in MSmedium (closed symbols) and modified MS-medium (open symbols), (A) OTR (hexagons) and ammonium concentration (diamonds), (B) wet (down triangles) and dry weight (circles), (C) HA (squares) and protein concentration (up triangles) related to cell broth. Experimental conditions: flask volume, 250 ml; filling volume, 50 ml; initial sucrose concentration, 30 g/l; temperature, 26°C; shaking frequency; 180 rpm; shaking diameter, 5 cm. Dotted lines represent the expected exhaustion of the ammonium concentration indicated by the bend in the OTR-curve. Due to an electrical power breakdown there was no data logging of the OTR between 41 h and 51 h.
of oxygen consumption has been proven to be a useful analytic tool, since changes of cell metabolism could be easily detected online. For the first time an online detection of the ammonium depletion based on the OTR is reported for a cultivation of N. tabacum BY-2 cells. A further analysis of the nutrients could show the depletion of ammonia as reason for that metabolic shift. Furthermore, this improved medium was successfully used for another transgenic tobacco cell line to characterize the growth behavior and to boost target product formation of the pharmaceutically relevant protein influenza hemagglutinin (HA). ACKNOWLEDGMENTS The authors gratefully thank Dr. Flora Schuster (RWTH Aachen University) for her excellent assistance with the plant tissue culture
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