Transgenic Rice Cell Suspension Cultures Research Articles

Production of Mature Recombinant Human Activin A in Transgenic Rice Cell Suspension Culture.

Activin A belongs to the transforming growth factor (TGF) family member, which exhibits a wide range of biological activities, including the regulation of cellular proliferation and differentiation and the promotion of neuronal survival. The isolation of AA from natural sources can only produce limited quantities of this bioactive protein. In this study, the whole gene of the precursor form of recombinant human activin A (rhAA) contains a signal peptide, and a pro-region and a mature region were cloned into an expression vector under the control of the rice α-amylase 3D (RAmy3D) promoter. To obtain the mature (active) form of rhAA, an enterokinase cleavage site was inserted between the pro-region and mature region of rhAA. The rice seed (Oryza sativa L. cv. Dongjin) was transformed with recombinant vectors by the Agrobacterium-mediated method, and the integration of the target gene into the plant genome was confirmed by genomic PCR. The transcript expression of rhAA in transgenic rice calli was confirmed by a Northern blot analysis of mRNA. The production of rhAA was verified by Western blot analysis and ELISA. The accumulation of secreted rhAA in the culture medium was purified by Ni2+-NTA. The mature form of AA was released from the precursor form of rhAA after proteolytically processing with enterokinase. Western blot shows that the mature AA was split into monomer and homodimer with molecular weights of 14 kDa and 28 kDa under reducing and non-reducing conditions, respectively. These results suggest that the mature form of rhAA could be produced and purified using transgenic rice cell suspension culture.

형질전환 벼 세포 배양에서 Sorbitol과 Hexokinase 저해제 조합 전략을 통한 Acid α-glucosidase 생산성 증대

Acid α-glucosidase (GAA) was produced in transgenic rice cell suspension cultures. In order to solve the problems using RAmy3D promoter for the efficient expression of GAA under sugar starvation condition, sorbitol was used as an alternative carbon source. Cell viability could be prolonged up to 27 days when sorbitol was present. Protein expression could also be enhanced. In addition, N-acetyl glucosamine, a hexokinase inhibitor, was used as a regulator for the RAmy3D promoter to shorten the incubation time with improved GAA expression. When sorbitol and N-acetyl glucosamine were used together, production of GAA was higher than that with sorbitol only. In order to optimize the combination condition, various experimental sets were designed and 60% increased protein could be obtained. In conclusion, optimized utilization of sorbitol as an alternative carbon source with N-acetyl glucosamine in transgenic rice cell suspension cultures was found to be the best condition for the enhanced expression of GAA using RAmy3D promoter.

Production of recombinant butyrylcholinesterase from transgenic rice cell suspension cultures in a pilot-scale bioreactor.

Producing recombinant proteins in transgenic plant cell suspension cultures in bioreactors provides controllability, reproducibility, scalability, and low-cost production, although low yields remain the major challenge. The studies on scaling-up to pilot-scale bioreactors, especially in conventional stainless-steel stirred tank bioreactors (STB), to produce recombinant proteins in plant cell suspension cultures are very limited. In this study, we scaled-up the production of rice recombinant butyrylcholinesterase (rrBChE), a complex hydrolase enzyme that can be used to prophylactically and therapeutically treat against organophosphorus nerve agents and pesticide exposure, from metabolically regulated transgenic rice cell suspension cultures in a 40-L pilot-scale STB. Employing cyclical operation together with a simplified-process operation (controlling gas sparging rate rather than dissolved oxygen and allowing natural sugar depletion) identified in lab-scale (5 L) bioreactor studies, we found a consistent maximum total active rrBChE production level of 46-58 µg/g fresh weight in four cycles over 82 days of semicontinuous operation. Additionally, maintaining the overall volumetric oxygen mass transfer coefficient (kL a) in the pilot-scale STB to be equivalent to the lab-scale STB improves the maximum total active rrBChE production level and the maximum volumetric productivity to 85 µg/g fresh weight and 387 µg L-1 day-1 , respectively, which are comparable to the lab-scale culture. Here, we demonstrate pilot-scale bioreactor performance using a metabolically regulated transgenic rice cell culture for long-term, reproducible, and sustained production of rrBChE.

Effects of Kifunensine on Production and N-Glycosylation Modification of Butyrylcholinesterase in a Transgenic Rice Cell Culture Bioreactor.

The production and N-glycosylation of recombinant human butyrylcholinesterase (BChE), a model highly glycosylated therapeutic protein, in a transgenic rice cell suspension culture treated with kifunensine, a strong α-mannosidase I inhibitor, was studied in a 5 L bioreactor. A media exchange was performed at day 7 of cultivation by removing spent sugar-rich medium (NB+S) and adding fresh sugar-free (NB-S) medium to induce the rice α-amylase 3D (RAmy3D) promoter to produce rice recombinant human BChE (rrBChE). Using a 1.25X-concentrated sugar-free medium together with an 80% reduced working volume during the media exchange led to a total active rrBChE production level of 79 ± 2 µg (g FW)−1 or 7.5 ± 0.4 mg L−1 in the presence of kifunensine, which was 1.5-times higher than our previous bioreactor runs using normal sugar-free (NB-S) media with no kifunensine treatment. Importantly, the amount of secreted active rrBChE in culture medium was enhanced in the presence of kifunensine, comprising 44% of the total active rrBChE at day 5 following induction. Coomassie-stained SDS-PAGE gel and Western blot analyses revealed different electrophoretic migration of purified rrBChE bands with and without kifunensine treatment, which was attributed to different N-glycoforms. N-Glycosylation analysis showed substantially increased oligomannose glycans (Man5/6/7/8) in rrBChE treated with kifunensine compared to controls. However, the mass-transfer limitation of kifunensine was likely the major reason for incomplete inhibition of α-mannosidase I in this bioreactor study.

Simplified bioreactor processes for recombinant butyrylcholinesterase production in transgenic rice cell suspension cultures

Metabolically regulated transgenic rice cell suspension culture has been developed aiming to be an efficient and cost-effective platform for production of rice-recombinant butyrylcholinesterase (rrBChE) that could be used as a bioscavenger enzyme against organophosphate nerve agents. In two-stage batch cultures, switching between growth and induction phases by exchanging media, the maximum active rrBChE levels were comparable among the bioreactor runs with controlled dissolved oxygen (DO) concentrations from 10–40% DO and without controlled DO at constant aeration rate. For a single-stage batch operation with no media exchange at 40% DO, production of rrBChE is triggered simply through sugar depletion due to the uptake of sugar by the cells, showing similar maximum active cell-associated rrBChE level compared to two-stage batch operations. However, the bioreactor process using uncontrolled DO, no media exchange, and half-concentration of sucrose resulted in a 1.4-fold increase in the maximum total active rrBChE (77 μg/g fresh weight) and 1.6-fold increase of total active rrBChE specific productivity (86 μg (g dry weight)−1 day−1) compared to the two-stage batch cultures. We found that monitoring the culture pH is a simple non-invasive technique that can help identify the start and progress of the induction phase in a single-stage culture. Altogether, we demonstrate a simplified, efficient, economical, and scalable bioreactor process for rrBChE production in metabolically regulated transgenic rice cell cultures.

Technoeconomic analysis of semicontinuous bioreactor production of biopharmaceuticals in transgenic rice cell suspension cultures.

Biopharmaceutical protein production using transgenic plant cell bioreactor processes offers advantages over microbial and mammalian cell culture platforms in its ability to produce complex biologics with simple chemically defined media and reduced biosafety concerns. A disadvantage of plant cells from a traditional batch bioprocessing perspective is their slow growth rate which has motivated us to develop semicontinuous and/or perfusion processes. Although the economic benefits of plant cell culture bioprocesses are often mentioned in the literature, to our knowledge no rigorous technoeconomic models or analyses have been published. Here we present technoeconomic models in SuperPro Designer® for the large-scale production of recombinant butyrylcholinesterase (BChE), a prophylactic/therapeutic bioscavenger against organophosphate nerve agent poisoning, in inducible transgenic rice cell suspension cultures. The base facility designed to produce 25 kg BChE per year utilizing two-stage semicontinuous bioreactor operation manufactures a single 400 mg dose of BChE for $263. Semicontinuous operation scenarios result in 4-11% reduction over traditional two-stage batch operation scenarios. In addition to providing a simulation tool that will be useful to the plant-made pharmaceutical community, the model also provides a computational framework that can be used for other semicontinuous or batch bioreactor-based processes.

Profiles of plant core-fucosylated N-glycans of acid alpha-glucosidases produced in transgenic rice cell suspension cultures treated with eight different conditions

Recombinant human acid alpha-glucosidase (rhGAA) from Chinese hamster ovary cells is the only approved treatment for patients with Pompe disease. In this study, rhGAAs were produced in transgenic rice cell suspension cultures under eight different conditions; untreated, 5 μM of 2-fluoro-l-fucose (2-FF), 50 μM of 2-FF, 100 μM of 2-FF, 100 μM of 2-FF + 0.5% Pluronic F-68 (PF-68), 100 μM of 2-FF + 0.05% Tween 20 (Tw 20), 0.5% PF-68, and 0.05% Tw 20. The N-glycans of eight rhGAAs were analyzed using ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry. The relative quantity (%) of each glycan was obtained from the corresponding UPLC peak area per the sum (100%) of individual UPLC peak area. Fifteen N-glycans, comprising seven core-fucosylated glycans (71.5%, sum of each relative quantities) that have immunogenicity-inducing potential, three de-core-fucosylated glycans (15.4%), and five non-core-fucosylated glycans (13.1%), were characterized with high mass accuracy and glycan-generated fragment ions. The increases or decreases of relative quantities of each glycan from seven rhGAAs were compared with those of untreated control. The percentages of the sum of the relative quantities of core-fucosylated glycans divided by the sums of those of de-core- (core-fucose removed) and non-core-fucosylated glycans were calculated, and the lowest percentage was obtained in 100 μM of 2-FF combined with 0.5% PF-68. These results indicate that the relative quantity of each glycan of rhGAA produced in rice cell suspension cultures is significantly affected by their culture condition. This study performed the comparison of the N-glycan profiles of rice cell-derived rhGAA to identify the core-fucosylated glycans using UPLC and tandem mass spectrometry.

Production of functional recombinant cyclic citrullinated peptide monoclonal antibody in transgenic rice cell suspension culture.

Cyclic citrullinated peptide (CCP) antibody has been shown recently to be a promising marker for early detection and diagnosis of rheumatoid arthritis (RA). In order to exploit newly developed therapies for RA, early intervention is crucial in preventing irreversible joint damage. Here, we describe use of a plant expression system to produce a CCP antibody that could be used in the early diagnosis of RA. Heavy and light chain gene sequences of a CCP monoclonal antibody (CCP mAb) were cloned from the hybridoma cell (12G1) and introduced into two separate plant expression vectors under the control of the rice α-amylase 3D (RAmy3D) promoter system. The vectors were introduced into rice calli (Oryza sativa L. cv. Dongjin) using Agrobacterium tumefaciens mediated transformation. Integration of the CCP mAb genes into rice chromosomes was confirmed by a genomic DNA polymerase chain reaction and expression was verified by northern blot analysis of mRNA. The in vivo assembly and secretion of CCP mAb occurred in transgenic rice cell suspension culture under the RAmy3D expression system; accumulated CCP mAbs in the medium were purified by protein G affinity chromatography. Immunoblot assays and ELISA showed these plant-produced CCP mAbs successfully bound to a synthetic CCP antigen. Taken together, our results suggest that CCP mAb produced in a transgenic rice suspension culture were easily purified and biologically active against their antigen in the RA, and thus may be used a specific serological marker, which is present very early in the RA.

Purification, characterization, and N-glycosylation of recombinant butyrylcholinesterase from transgenic rice cell suspension cultures.

Recombinant butyrylcholinesterase produced in a metabolically regulated transgenic rice cell culture (rrBChE) was purified to produce a highly pure (95%), active form of enzyme. The developed downstream process uses common manufacturing friendly operations including tangential flow filtration, anion-exchange chromatography, and affinity chromatography to obtain a process recovery of 42% active rrBChE. The purified rrBChE was then characterized to confirm its comparability to the native human form of the molecule (hBChE). The recombinant and native enzyme demonstrated comparable enzymatic behavior and had an identical amino acid sequence. However, rrBChE differs in that it contains plant-type complex N-glycans, including an α-1,3 linked core fucose, and a β-1,2 xylose, and lacking a terminal sialic acid. Despite this difference, rrBChE is demonstrated to be an effective stoichiometric bioscavenger for five different organophosphorous nerve agents in vitro. Together, the efficient downstream processing scheme and functionality of rrBChE confirm its promise as a cost-effective alternative to hBChE for prophylactic and therapeutic use.

Spontaneous pepsin C-catalyzed activation of human pepsinogen C in transgenic rice cell suspension culture: Production and characterization of human pepsin C.

A human pepsinogen C (hPGC) gene was synthesized with rice-optimized codon usage and cloned into a rice expression vector containing the promoter, signal peptide, and terminator derived from the rice α-amylase 3D (Ramy3D) gene. In addition, a 6-His tag was added to the 3' end of the synthetic hPGC gene for easy purification. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin) mediated by Agrobacterium tumefaciens. The integration of the hPGC gene into the chromosome of the transgenic rice callus and hPGC expression in transgenic rice cell suspensions was verified via genomic DNA polymerase chain reaction amplification and Northern blot analysis. Western blot analysis indicated both hPGC and its mature form, human pepsin C, with masses of 42- and 36-kDa in the culture medium under sugar starvation conditions. Human pepsin C was purified from the culture medium using a Ni-NTA agarose column and the NH2-terminal 5-residue sequences were verified by amino acid sequencing. The hydrolyzing activity of human pepsin C was confirmed using bovine hemoglobin as a substrate. The optimum pH and temperature for pepsin activity were 2.0 and 40°C, respectively.

Production of monoclonal antibodies against 53-kDa protein of Porphyromonas gingivalis in transgenic rice cell suspension culture

Periodontitis is a bacterial infection-induced inflammatory disease that affects the tissues surrounding the teeth and is the main cause of tooth loss in adults. Porphyromonas gingivalis, a black-pigmented gram-negative anaerobe, is a major causative agent of periodontitis. A 53-kDa protein of the bacterium has been demonstrated to be highly immunogenic and recognized by sera from most periodontitis patients, and it is now considered as a potential target of protective immunity. In this study, complementary DNA encoding monoclonal antibodies to 53-kDa protein of P. gingivalis A7A1-28 was cloned from hybridoma cell lines expressing the antibodies. The heavy and light chain genes were constructed into plant expression vectors under the control of the rice α-amylase 3D (RAmy3D) promoter. The recombinant plant expression vectors were introduced into rice cells (Oryza sativa L. cv. Dongjin) using particle bombardment transformation method. Transgenic rice calli were selected by genomic DNA PCR and used to establish cell suspension cultures. The expression of the heavy and light chain genes of the monoclonal antibodies in the rice cell suspension cultures was detected by western blot analysis. The plant-produced monoclonal antibodies showed specific reactivity with 53-kDa proteins of P. gingivalis. These results suggest that 53-kDa protein-specific monoclonal antibodies produced in the rice cell suspension cultures are biologically active against the bacterial protein and can be used for passive immunization to prevent P. gingivalis-induced periodontal disease.

Production and characterization of recombinant human acid α-glucosidase in transgenic rice cell suspension culture

Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid α-glucosidase (GAA), a glycogen-degrading lysosomal enzyme. In this study, the human GAA cDNA gene was synthesized from human placenta cells and cloned into a plant expression vector under the control of the rice α-amylase 3D (RAmy3D) promoter. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin) mediated by Agrobacterium tumefaciens. Genomic DNA PCR and Northern blot analysis were used to determine the integration and mRNA expression of the hGAA gene in the putative transgenic rice cells. SDS-PAGE and Western blot analysis showed that the glycosylated precursor recombinant hGAA had a molecular mass of 110kDa due to the presence of seven N-glycosylation sites. The accumulation of hGAA protein in the culture medium was approximately 37mg/L after 11 days of culturing in a sugar depletion medium. The His tagged-hGAA protein was purified using an Ni-NTA column and confirmed as the precursor form of hGAA without the signal peptide encoded by the cDNA on the N-terminal amino acid sequence. The acid alpha-glucosidase activity of hGAA produced in transgenic rice cells gave results similar to those of the enzyme produced by CHO cells.

Semicontinuous Bioreactor Production of Recombinant Butyrylcholinesterase in Transgenic Rice Cell Suspension Cultures.

An active and tetrameric form of recombinant butyrylcholinesterase (BChE), a large and complex human enzyme, was produced via semicontinuous operation in a transgenic rice cell suspension culture. After transformation of rice callus and screening of transformants, the cultures were scaled up from culture flask to a lab scale bioreactor. The bioreactor was operated through two phases each of growth and expression. The cells were able to produce BChE during both expression phases, with a maximum yield of 1.6 mg BChE/L of culture during the second expression phase. Cells successfully regrew during a 5-day growth phase. A combination of activity assays and Western blot analysis indicated production of an active and fully assembled tetramer of BChE.

Production of functional human vascular endothelial growth factor165 in transgenic rice cell suspension cultures

Vascular endothelial growth factors (VEGFs) are secreted by tumor cells and other cells exposed to hypoxia, and play a critical role in the development and differentiation of the vascular system. In this study, we investigated the production of functional recombinant human VEGF165 (rhVEGF165) in transgenic rice cell suspension culture. Complementary DNA was synthesized from human leukemia HL60 cells and cloned into expression vectors under the control of the rice α-amylase 3D (RAmy3D) promoter. The rice seed (Oryza sativa L. cv. Dongjin) was transformed with this recombinant vector by the Agrobacterium mediated method and the integration of the target gene into the plant genome was confirmed by genomic PCR. The expression of rhVEGF165 in the rice cells was determined by Northern blot and Western blot analyses. The accumulated rhVEGF165 protein in the culture medium was 19mg/L after 18 days of culturing in a sugar-free medium. The rhVEGF165 was purified using a heparin HP column and its biological activity was tested on human umbilical vein endothelial cells (HUVECs). The purified rhVEGF165 significantly increased the proliferative activity of the HUVECs. Therefore, it was demonstrated that functional rhVEGF165 could be produced using transgenic rice suspension culture vector under the control of the RAmy3D promoter.

High-level production of recombinant trypsin in transgenic rice cell culture through utilization of an alternative carbon source and recycling system

Productivity of recombinant bovine trypsin using a rice amylase 3D promoter has been studied in transgenic rice suspension culture. Alternative carbon sources were added to rice cell suspension cultures in order to improve the production of recombinant bovine trypsin. It was demonstrated that addition of alternative carbon sources such as succinic acid, fumaric acid and malic acid in the culture medium could increase the productivity of recombinant bovine trypsin 3.8–4.3-fold compared to those in the control medium without carbon sources. The highest accumulated trypsin reached 68.2mg/L on day 5 in the culture medium with 40mM fumaric acid.The feasibility of repeated use of the cells for recombinant trypsin production was tested in transgenic rice cell suspension culture with the culture medium containing the combination of variable sucrose concentration and 40mM fumaric acid. Among the used combinations, the combination of 1% sucrose and 40mM fumaric acid resulted in a yield of up to 53mg/L five days after incubation. It also increased 31% (W/W) of dry cell weight and improved 43% of cell viability compared to that in control medium without sucrose. Based on these data, recycling of the trypsin production process with repeated 1% sucrose and 40mM fumaric acid supplying-harvesting cycles was developed in flask scale culture. Recombinant bovine trypsin could be stably produced with a yield of up to 53–39mg/L per cycle during five recycling cycles.

Assessment of long-term cryopreservation for production of hCTLA4Ig in transgenic rice cell suspension cultures

For the commercialization of plant-made pharmaceuticals (PMPs) using transgenic plant cell cultures, the establishment of a cell-banking system has been known to be an essential process. Plant cells are traditionally maintained by repeated subcultures. However, this method has several problems including genetic instability of transformed cell lines, time- and cost-consuming. In this study, long-term cryopreserved rice suspension cells were firstly investigated for the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig). The cryopreserved cells for 5 years were regrowed to callus successfully and then suspended into the liquid medium. Consequently, the maximum cell mass and the hCTLA4Ig production were similar levels compared to those of the non-cryopreserved cells (control) even though hCTLA4Ig productivity was 1.7-fold higher than that of control. To further assess the level of improvements in hCTLA4Ig productivity in cryopreserved cells, hCTLA4Ig production profiles were statistically assessed between data of the cryopreserved cells for 5 years and annual data of non-cryopreserved cells maintained by subculture for 5 years. These results also indicate that hCTLA4Ig productivity in cryopreserved cells for 5 years was significantly increased (p-value: <0.001, 95% confidence interval) and it could be related to cell lysis resulting in release of hCTLA4Ig which was confirmed by the measurement of electrolyte leakage. In conclusion, we show that the long-term cryopreservation of transgenic rice cells was possible to support stable cell lines for the production of PMPs.

Amylase and cysteine proteinase gene knockdown in rice cells using RNA interference for enhancing production of recombinant proteins

A rice cell suspension culture with the rice α-amylase 3D promoter expression system which is induced by sucrose starvation was previously reported to generate a good yield of recombinant proteins. However, this expression system is limited by the accumulation of undesirable α-amylase and proteases in the culture medium. Rice α-amylase is a dominant protein at 43 % of total secreted proteins, and cysteine proteinase (CysP) is a major secreted protease in rice cell suspension cultures following induction via sugar depletion. Here, we nearly eliminated rice α-amylase and CysP proteinase via RNA interference (RNAi) technology to improve the recombinant protein yield in rice cell suspension culture. The effects of RNAi were characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Western blot analysis with anti-CysP antibody, and quantitative real-time reverse transcription polymerase chain reaction analysis (RT-PCR). The mRNA levels of α-amylase and CysP were reduced by 94.8 and 95.0 %, respectively. Transgenic rice cell suspension cultures expressing both human granulocyte–macrophage colony-stimulating factor (hGM-CSF) and ihpRNA of α-amylase and CysP genes evidenced a reduction of α-amylase and CysP activity and up to 2.4-fold improvement of hGM-CSF production compared to that in a transgenic cell line expressing hGM-CSF only. Our rice cell suspension culture for the reduction of α-amylase accumulation and protease activity in the culture medium could improve recombinant protein production as an efficient protein expression system using RNA interference technology in plant biotechnology.

Adsorptive loss of secreted recombinant proteins in transgenic rice cell suspension cultures

Adsorptive loss of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) in transgenic rice cell suspension cultures was investigated using glass flasks, plastic flasks, disposable vessels, and stainless steel vessels. When hCTLA4Ig was added to the glass flasks containing sterile AA medium, a rapid decrease in the concentration of hCTLA4Ig, independent on pH, was observed resulting in more than 90% of the protein loss within 1 h due to the surface adsorption. When the same experiments were performed on four different types of culture equipments mentioned above, the lowest adsorption level was observed in the plastic flasks and the highest level was observed in the glass flasks. The use of the plastic flasks retarded the adsorptive loss of hCTLA4Ig at the early stage of the protein production. There was a significant increase in the production of hCTLA4Ig when the flasks were coated with bovine serum albumin. However, the spike test of purified hCTLA4Ig at two different concentrations of 15 and 100 mg L(-1) in 500-mL spinner flasks confirmed that the amount of hCTLA4Ig adsorbed was dependent on the surface area of the flasks but not on the concentrations. In conclusion, although the protein adsorption affected the total amount of the protein yielded to some extent, it could be regarded as a minor factor in transgenic plant cell cultures with higher titer.

Sodium butyrate와 sodium pyruvate 첨가에 의한 hCTLA4Ig 생산성 증대

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig), an immunosuppressive agent, was expressed in transgenic rice cells using RAmy3D promoter and RAmy1A signal peptide for the inducible production and secretion into culture media by sugar depletion. In this study, sodium butyrate was used as a small molecular enhancer (SME) to enhance the production of hCTLA4Ig in transgenic rice cell suspension cultures. When 1 mM sodium butyrate was added in sugar-free media, relative viability was not reduced, while the productivity was improved 1.3-fold. In addition, by supplementing 87 mM sodium pyruvate as an alternative energy source during the production phase, death rate of the cells was decreased. When sodium pyruvate was not added, most cells became dead at day 6. However, by adding sodium pyruvate, 18% of viability can be maintained until day 10 and the production of hCTLA4Ig was enhanced 1.4-fold. When the combination of sodium pyruvate and sodium butyrate at optimum concentrations was added, the highest viability and hCTLA4Ig production could be obtained. The highest level of hCTLA4Ig reached up to 35 mg/L at day 10.

Production of functional recombinant bovine trypsin in transgenic rice cell suspension cultures

A synthetic bovine trypsinogen (sbTrypsinogen) was synthesized on the basis of rice-optimized codon usage via an overlap PCR strategy, prior to being expressed under the control of the sucrose starvation-inducible rice α-amylase 3D (RAmy3D) promoter. Secretion of trypsin into the culture medium was achieved by using the existing signal peptide. The plant expression vector was introduced into rice calli ( Oryza sativa L. cv. Dongjin), mediated by Agrobacterium tumefaciens. The integration of the sbTrypsinogen gene into the chromosome of the transgenic rice callus was verified via genomic DNA PCR amplification, and sbTrypsin expression in transgenic rice suspension cells was confirmed via Northern blot analysis. Western blot analysis detected glycosylated proteins in the culture medium, having masses from 24 to 26 kDa, following induction by sugar starvation. Proteolytic activity of the rice-derived trypsin was confirmed by gelatin zymogram, and was similar to that of the commercial bovine-produced trypsin. The yields of sbTrypsin that accumulated in the transgenic rice cell suspension medium were 15 mg/L at 5 days after sugar starvation.