Rice Cell Suspension Research Articles

H₂O₂ Is Involved in the Metallothionein-Mediated Rice Tolerance to Copper and Cadmium Toxicity.

Cadmium (Cd) and excess copper (Cu) are toxic to plants, causing a wide range of deleterious effects including the formation of reactive oxygen species. Metallothioneins (MTs) may protect plant cells from heavy metal toxicity by chelating heavy metals via cysteine thiol groups. They may also function as antioxidants. The study investigated the relationship of H2O2 production and ricMT expression in rice radicles and rice suspension cells under Cu or Cd stress. The results showed that H2O2 production in the rice radicles increased before Cu-induced ricMT expression, and after Cd-induced ricMT expression. Rice suspension cells of sense- and antisense-ricMT transgenic lines were obtained by an Agrobacterium-mediated transformation. Overexpression of ricMT significantly decreased the death rate of rice cells, which was accompanied by blocked H2O2 accumulation in rice suspension cells subject to Cu and Cd stress. Our findings confirm that H2O2 is involved in the MT-mediated tolerance of Cu and Cd toxicity in rice.

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.

The cyclic lipopeptide orfamide induces systemic resistance in rice to Cochliobolus miyabeanus but not to Magnaporthe oryzae.

The Pseudomonas- derived cyclic lipopeptide orfamide can induce resistance to Cochliobolus miyabeanus but not to Magnaporthe oryzae in rice. Abscisic acid signaling is involved in the induced systemic resistance response triggered by orfamide. Diverse natural products produced by beneficial Pseudomonas species have the potential to trigger induced systemic resistance (ISR) in plants, and thus may contribute to control of diseases in crops. Some beneficial Pseudomonas spp. can produce cyclic lipopeptides (CLPs), amphiphilic molecules composed of a fatty acid tail linked to an oligopeptide which is cyclized. CLPs can have versatile biological functions, but the capacity of Pseudomonas-derived CLPs in triggering ISR responses has barely been studied. Pseudomonas protegens and related species can produce orfamide-type CLPs. Here we show that in rice, orfamides can act as ISR elicitors against the necrotrophic fungus Cochliobolus miyabeanus, the causal agent of brown spot disease, but are not active against the blast fungus Magnaporthe oryzae. Orfamide A can trigger early defensive events and activate transcripts of defense-related genes in rice cell suspension cultures, but does not cause cell death. Further testing in rice cell suspension cultures and rice plants showed that abscisic acid signaling, the transcriptional activator OsWRKY4 and pathogenesis-related protein PR1b are triggered by orfamide A and may play a role in the ISR response against C. miyabeanus.

Synergistic effects between [Si-hemicellulose matrix] ligands and Zn ions in inhibiting Cd ion uptake in rice (Oryza sativa) cells.

Our study demonstrated that Zn alleviated Cd toxicity in the presence of Si in the cell walls by Zn 2+ binding to ligands through the formation of the [Si-hemicellulose matrix]Zn complexes that restrict the uptake of Cd. The plant cell wall exhibits preferential sites for the accumulation of metals at toxic concentrations. Through modification of wall polysaccharide components, elements, such as silicon (Si) and zinc (Zn), may play active roles in alleviating the toxicity of heavy metals, including cadmium (Cd). However, enhanced tolerance for Cd stress may rely on synergistic effects between nutrient elements. Here, we cultured Si-accumulating suspension cells of rice (Oryza sativa) exposed to Cd and Zn treatments, either separately or in combination, and investigated cells using noninvasive microtest technology (NMT), inductively coupled plasma mass spectroscopy (ICP-MS) and atomic force microscopy (AFM). We found that Zn alleviated Cd toxicity in the presence of Si in the cell walls by binding of Zn2+ to ligands through the formation of the [Si-hemicellulose matrix]Zn complexes and co-precipitates to greatly inhibit Cd2+ uptake into cells. This, in turn, induced the lower expression of Cd-related transporters. This synergistic effect could be decisive for the survival of cells under conditions of high Cd concentrations.

Exogenous ATP enhance signal response of suspension cells of transgenic rice (Oryza sativa L.) expressing maize C 4 -pepc encoded phosphoenolpyruvate carboxylase under PEG treatment

The aim of this study was to analyze the biochemical and molecular responses of suspension cells of transgenic rice (Oryza sativa L.) over-expressing the maize C 4 -pepc gene encodes phosphoenolpyruvate carboxylase (PEPC) (PC line) to drought stress by exogenous ATP. Suspension cells of PC and wild-type (WT) rice cultured in N6 basic medium were subjected to the following treatments: a 24-h drought treatment imposed by 20% (m/v) polyethylene glycol 6000 (PEG6000 treatment); an exogenous adenosine 5′-triphosphate treatment (ATP; 1 mmol L−1); and a combined exogenous ATP + PEG6000 treatment. We measured DNA fragmentation, cell viability, lipid peroxidation, antioxidant enzyme activities, PEPC activity, ATP content, and the levels of Ca2+, H2O2, and NO using spectrophotometric methods and fluorescent dyes, and quantified the transcript levels of stress-related genes such as NAC1 (one of the transcription factors from NAC (NAM/ATAF/CUC) family), cytochrome P450 (P450) and programmed cell death (PCD) under these treatments. Compared with WT cells, PC cells showed more stable cellular activity, lower lipid peroxidation, enhanced antioxidant enzyme activity, higher levels of endogenous ATP level, PEPC activity, and transcript levels of C 4 -pepc and the transcription factor NAC1 under drought stress. While the content of H2O2 and the transcript levels of P450 and PCD were decreased and kept at the low levels during the same treatments. Adding ATP, PC reinforced these early signaling effects response to PEG6000 treatment. Based on the results, the exogenous ATP triggers a series of signaling cascade reactions for enhancing antioxidant protection in PC cells during the drought treatment, conferring to the drought-tolerance.

Enrichment of the Plant Cytosolic Fraction.

The cytosol is at the core of cellular metabolism and contains many important metabolic pathways, including glycolysis, gluconeogenesis, and the pentose phosphate pathway. Despite the importance of this matrix, few attempts have sought to specifically enrich this compartment from plants. Although a variety of biochemical pathways and signaling cascades pass through the cytosol, much of the focus has usually been targeted at the reactions that occur within membrane-bound organelles of the plant cell. In this chapter, we outline a method for the enrichment of the cytosol from rice suspension cell cultures which includes sample preparation and enrichment as well as validation using immunoblotting and fluorescence-tagged proteins.

Glucosidase II β-subunit, a novel substrate for caspase-3-like activity in rice, plays as a molecular switch between autophagy and programmed cell death.

Endoplasmic reticulum (ER) stress activates unfolded protein response (UPR) and autophagy. However, prolonged, severe stresses activate programmed cell death (PCD) in both animal and plant cells. Compared to the well-studied UPR pathway, the molecular mechanisms of ER-stress-induced PCD are less understood. Here, we report the identification of Gas2, the glucosidase II β subunit in the ER, as a potential switch between PCD and autophagy in rice. MS analysis identified Gas2, GRP94, and HSP40 protein in a purified caspase-3-like activity from heat stressed rice cell suspensions. The three corresponding genes were down-regulated under DTT-induced ER stress. Gas2 and GRP94 were localized to the ER, while HSP40 localized to the cytoplasm. Compared to wild-type, a Gas2 RNAi cell line was much sensitive to DTT treatment and had high levels of autophagy. Both caspase-3 and heat-stressed cell suspension lysate could cleave Gas2, producing a 14 kDa N-terminal fragment. Conditional expression of corresponding C-terminal fragment resulted in enhanced caspase-3-like activity in the protoplasts under heat stress. We proposed that mild ER stress causes down-regulation of Gas2 and induces autophagy, while severe stress results in Gas2 cleavage by caspase-3-like activity and the cleavage product amplifies this activity, possibly participating in the initiation of PCD.

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.

Fate and metabolism of the brominated flame retardant tetrabromobisphenol A (TBBPA) in rice cell suspension culture

Tetrabromobisphenol A (TBBPA) is the brominated flame retardant with the highest production volume and its bioaccumulation in environment has caused both human health and environmental concerns, however the fate and metabolism of TBBPA in plants is unknown. We studied the fate, metabolites, and transformation of 14C-labeled TBBPA in rice cell suspension culture. During the incubation for 14 days, TBBPA degradation occurred continuously in the culture, accompanied by formation of one anisolic metabolite [2,6-dibromo-4-(2-(2-hydroxy)-propyl)-anisole] (DBHPA) (50% of the degraded TBBPA) and cellular debris-bound residues (46.4%) as well as mineralization (3.6%). The cells continuously accumulated TBBPA in the cytoplasm, while a small amount of DBHPA (2.1% of the initially applied TBBPA) was detectable inside the cells only at the end of incubation. The majority of the accumulated residues in the cells was attributed to the cellular debris-bound residues, accounting for 70–79% of the accumulation after the first incubation day. About 5.4% of the accumulation was associated with cell organelles, which contributed 7.5% to the cellular debris-bound residues. Based on the fate and metabolism of TBBPA in the rice cell suspension culture, a type II ipso-substitution pathway was proposed to describe the initial step for TBBPA degradation in the culture and balance the fate of TBBPA in the cells. To the best of our knowledge, our study provides for the first time the insights into the fate and metabolism of TBBPA in plants and points out the potential role of type II ipso-hydroxylation substitution in degradation of alkylphenols in plants. Further studies are required to reveal the mechanisms for the bound-residue formation (e.g., binding of residues to specific cell wall components), nature of the binding, and toxicological effects of the bound residues and DBHPA.

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.

ITRAQ-based proteomic analysis reveals the mechanisms of silicon-mediated cadmium tolerance in rice (Oryza sativa) cells

Silicon (Si) can alleviate cadmium (Cd) stress in rice (Oryza sativa) plants, however, the understanding of the molecular mechanisms at the single-cell level remains limited. To address these questions, we investigated suspension cells of rice cultured in the dark environment in the absence and presence of Si with either short- (12 h) or long-term (5 d) Cd treatments using a combination of isobaric tags for relative and absolute quantitation (iTRAQ), fluorescent staining, and inductively coupled plasma mass spectroscopy (ICP-MS). We identified 100 proteins differentially regulated by Si under the short- or long-term Cd stress. 70% of these proteins were down-regulated, suggesting that Si may improve protein use efficiency by maintaining cells in the normal physiological status. Furthermore, we showed two different mechanisms for Si-mediated Cd tolerance. Under the short-term Cd stress, the Si-modified cell walls inhibited the uptake of Cd ions into cells and consequently reduced the expressions of glycosidase, cell surface non-specific lipid-transfer proteins (nsLTPs), and several stress-related proteins. Under the long-term Cd stress, the amount of Cd in the cytoplasm in Si-accumulating (+Si) cells was decreased by compartmentation of Cd into vacuoles, thus leading to a lower expression of glutathione S-transferases (GST). These results provide protein-level insights into the Si-mediated Cd detoxification in rice single cells.

Potent In Vitro and In Vivo Activity of Plantibody Specific for Porphyromonas gingivalis FimA

Fimbrial protein fimbrillin (FimA), a major structural subunit of Porphyromonas gingivalis, has been suggested as a vaccine candidate to control P. gingivalis-induced periodontal disease. Previously, cDNAs encoding IgG monoclonal antibodies (MAbs) against purified FimA from P. gingivalis 2561 have been cloned, and the MAbs have been produced in rice cell suspension. Here we examined the biological activities of the plant-produced MAb specific for FimA (anti-FimA plantibody) of P. gingivalis in vitro and in vivo. The anti-FimA plantibody recognized oligomeric/polymeric forms of native FimA in immunoblot analysis and showed high affinity for native FimA (KD = 0.11 nM). Binding of P. gingivalis (10(8) cells) to 2 mg of saliva-coated hydroxyapatite beads was reduced by 53.8% in the presence of 1 μg/ml plantibody. Anti-FimA plantibody (10 μg/ml) reduced invasion of periodontal ligament cells by P. gingivalis (multiplicity of infection, 100) by 68.3%. Intracellular killing of P. gingivalis opsonized with the anti-FimA plantibody by mouse macrophages was significantly increased (77.1%) compared to killing of bacterial cells with irrelevant IgG (36.7%). In a mouse subcutaneous chamber model, the number of recoverable P. gingivalis cells from the chamber fluid was significantly reduced when the numbers of bacterial cells opsonized with anti-FimA plantibody were compared with the numbers of bacterial cells with irrelevant IgG, 66.7% and 37.1%, respectively. These in vitro and in vivo effects of anti-FimA plantibody were comparable to those of the parental MAb. Further studies with P. gingivalis strains with different types of fimbriae are needed to investigate the usefulness of anti-FimA plantibody for passive immunization to control P. gingivalis-induced periodontal disease.

Production of bioactive chitosan oligosaccharides using the hypertransglycosylating chitinase-D from Serratia proteamaculans

The biological activities of chitosan and its oligosaccharides are greatly influenced by properties such as the degree of polymerization (DP), degree of acetylation (DA) and pattern of acetylation (PA). Here, structurally diverse chitosan oligosaccharides from chitosan polymers (DA=35% or 61%) were generated using Serratia proteamaculans wild-type chitinase D (SpChiD) and the W114A mutant which lacks transglycosylase activity. The crude oligosaccharide mixtures and purified fractions with specific DP and DA ranges were tested for their ability to induce an oxidative burst in rice cell suspension cultures. The crude mixtures were more active when produced by the W114A mutant whereas the purified fractions were more active when produced by wild-type SpChiD. Neither hydrolysis nor transglycosylation by SpChiD was inhibited in the presence of fully-deacetylated oligosaccharides, suggesting that SpChiD could be exploited to generate oligosaccharides with defined DA and PA values.

Enhancement of recombinant human serum albumin in transgenic rice cell culture system by cultivation strategy

Fusion of the sugar-starvation-induced αAmy3 promoter with its signal peptide has enabled secretion of recombinant human serum albumin (rHSA) into the culture medium. To simplify the production process and increase the rHSA yield in rice suspension cells, a one-step strategem without medium change was adopted. The yield of rHSA was increased sixfold by this one-step approach compared with the two-step recombinant protein process, in which a change of the culture medium to sugar-free medium is required. The one-step strategem was applied to check repeated cycle of rHSA production, and the production of rHSA was also higher in each cycle in the one-step, as opposed to the two-step, production process. The use of the one-step process resulted in fewer damaged cells during the cell sugar starvation phase for recombinant protein production. Furthermore, we scaled up the rHSA production in a 2-L airlift and a 2-L stirred tank bioreactor by the one-step approach, and concluded that rHSA can be enriched to 45 mg L(-1) in plant culture commonly used MS medium by the airlift-type bioreactor. Our results suggest that rHSA production can be enriched by this optimized cultivation strategem.

Do rice suspension-cultured cells treated with abscisic acid mimic developing seeds?

Starch synthesis is activated in the endosperm during seed development and also in rice suspension cells cultured with abscisic acid. In the anticipation that the mechanisms of starch synthesis are similar between the endosperm and the suspension cells cultured with abscisic acid, expression of genes involved in starch synthesis was evaluated in the suspension cells after abscisic acid treatment. However, it was found that the regulatory mechanism of starch synthesis in the suspension cells cultured with abscisic acid was different from that in developing seeds. Expression analyses of genes involved in oil bodies, which accumulate in the embryo and aleurone layer, and seed storage proteins, which accumulate mainly in the endosperm, showed that the former were activated in the suspension cells cultured with abscisic acid, but the latter were not. Master regulators for embryogenesis, OsVP1 (homologue of AtABI3) and OsLFL1 (homologue of AtFUS3 or AtLFL2), were expressed in the suspension cells at levels comparable to those in the embryo. From these results, it is suggested that interactions between regulators and abscisic acid control the synthesis of phytic acid and oil bodies in the cultured cells and embryo. We suggest that the system of suspension cells cultured with abscisic acid helps to reveal the mechanisms of phytic acid and oil body synthesis in embryo.

The B″ regulatory subunit of protein phosphatase 2A mediates the dephosphorylation of rice retinoblastoma-related protein-1.

The phosphorylation of plant retinoblastoma-related (RBR) proteins by cyclin-dependent kinases (CDKs) is well documented, but the counteracting phosphatases have not been identified yet. We report here that rice retinoblastoma-related protein-1 (OsRBR1) interacted with the B″ subunit of rice protein phosphatase 2A (OsPP2A B″) and underwent reversible phosphorylation during the cell division cycle. The OsRBR1-OsPP2A B" association required B domain in OsRBR1 and the C-terminal region of OsPP2A B″. We found by immunoprecipitation that OsPP2A B″, OsPP2A catalytic subunit subtype II, PSTAIRE-type CDK and OsRBR1 were in the same protein complex, indicating a physical association between the phosphatase, the kinase and their common substrate. OsPP2A B″ contains three predicted CDK phosphorylation sites: Ser95, Ser102 and Ser119. The in vitro phosphorylation of Ser95 and Ser119 with PSTAIRE-kinases was verified by mass spectrometry. We generated a series of phosphorylation site mutants to mimic the dephosphorylated or phosphorylated states of OsPP2A B″, and confirmed that all of the three predicted sites can be phosphorylated. Yeast two-hybrid experiments suggested that the phosphorylation of OsPP2A B″ promoted the formation of the OsPP2A holoenzyme. A triple phosphorylation mimicking OsPP2A B″ mutant containing holoenzyme showed higher activity in phosphatase assays. Our data collectively show that the phosphatase activity of OsPP2A against OsRBR1 is regulated by the phosphorylation of its B″ regulatory subunit. However, the analysis of the effect of okadaic acid, a phosphatase inhibitor, in rice cell suspension cultures revealed that the dephosphorylation of OsRBR1 was completely inhibited only by high dose (300nM) of the okadaic acid during the cell cycle progression. Therefore the role of the protein phosphatase 1 should be considered as an additional post translational regulatory component of RBR protein function in higher plants.

Phosphohexose mutase of Xanthomonas oryzae pv. oryzicola is negatively regulated by HrpG and HrpX, and required for the full virulence in rice

The genome of Xanthomonas oryzae pv. oryzicola annotates one uncharacterized gene, XOC_3841, only one ORF in this strain is annotated to encode Phosphohexose mutase (XanA), which reversibly converts glucose 1-phosphate to glucose 6-phosphate that implicates in the carbon metabolism pathways. However, it is unclear whether the XanA-coding gene is involved in the full virulence of X. oryzae pv. oryzicola. In this report, we showed that the mutagenesis in unique xanA, led the pathogen effectively to unable to utilize glucose and galactose for growth. The expression of xanA was strongly induced by glucose, sucrose, fructose, mannose or galactose at least 3 times higher than that by non-sugar NY medium. Intriguingly, xanA promoter region contains an imperfect PIP-box (plant-inducible promoter) (TTCGC-N16-TTCGA), and the expression of xanA was inducible in rice suspension cells rather than in a nutrient-rich (NB) medium and negatively regulated by a key hrp regulatory HrpG and HrpX cascade. More importantly, mutation in xanA resulted in impairment of bacterial growth and virulence in planta, and reduced bacterial cell motility and extracellular polysaccharides (EPS) production in media. In addition, the lost properties mentioned above in RΔxanA were completely restored to the wild-type level by the presence of xanA in trans. All these results suggest that xanA is required for EPS production, cell motility and the full virulence of X. oryzae pv. oryzicola.

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.