Production of functional human α1-antitrypsin by plant cell culture

Recombinant human α₁-antitrypsin (rAAT) was expressed and secreted from transgenic rice cell suspension cultures in its biologically active form. This was accomplished by transforming rice callus tissues with an expression vector, p3D-AAT, containing the cDNA for mature human AAT protein. Regulated expression and secretion of rAAT from this vector was achieved using the promoter, signal peptide, and terminator from a rice α-amylase gene Amy3D. The Amy3D gene of rice is tightly controlled by simple sugars such as sucrose. It was possible, therefore, to induce the expression of the rAAT by removing sucrose from the cultured media or by allowing the rice suspension cells to deplete sucrose catabolically. Although transgenic rice cell produced a heterogeneous population of the rAAT molecules, they had the same N-terminal amino acids as those found in serum-derived (native) AAT from humans. This result indicates that the rice signal peptidase recognizes and cleaves the novel sequence between the Amy3D signal peptide and the first amino acid of the mature human AAT. The highest molecular weight band seen on Western blots (AAT top band) was found to have the correct C-terminal amino acid sequence and normal elastase binding activity. Staining with biotin-concanavalin A and avidin horseradish peroxidase confirmed the glycosylation of the rAAT, albeit to a lesser extent than that observed with native AAT. The rAAT, purified by immunoaffinity chromatography, had the same association rate constant for porcine pancreatic elastase as the native AAT. Thermostability studies revealed that the rAAT and native AAT decayed at the same rate, suggesting that the rAAT is correctly folded. The productivity of rice suspension cells expressing rAAT was 4.6–5.7 mg/g dry cell. Taken together, these results support the use of rice cell culture as a promising new expression system for production of biologically active recombinant proteins. Received: 18 January 1999 / Received revision: 26 April 1999 / Accepted: 1 May 1999     

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

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

Characterization of an immunomodulatory Der p 2-FIP-fve fusion protein produced in transformed rice suspension cell culture

Der p 2, a major allergen of Dermatophagoides pteronyssinus mites, is one of the most clinically relevant allergens to allergic patients worldwide. FIP-fve protein (Fve) from the golden needle mushroom (Flammulina velutipes) is an immunomodulatory protein with potential Th1-skewed adjuvant properties. Here, we produced and immunologically evaluated a Der p 2-Fve fusion protein as a potential immunotherapeutic for allergic diseases. Using an inducible expression system in cultured rice suspension cells, the recombinant Der p 2-Fve fusion protein (designated as OsDp2Fve) was expressed in rice cells under the control of an α-amylase gene (αAmy8) promoter and secreted under sucrose starvation. OsDp2Fve was partially purified from the cultured medium. The conformation of Der p 2 in OsDp2Fve remains intact as reflected by its unaltered allergenicity, as assessed by human IgE ELISA and histamine release assays, compared to non-fusion Der p 2 protein. Furthermore, the Fve protein expressed in OsDp2Fve retains its in vitro lymphoproliferative activity but loses its hemagglutination and lymphoagglutination effects compared to the native protein. Notably, in vivo evaluation showed that mice administered with OsDp2Fve possessed an enhanced production of Der p 2-specific IgG antibodies without potentiating the production of Der p 2-specific IgE and Th2 effector cytokines in comparison with mice co-administered with native Fve and Der p 2 proteins. These results suggest that the recombinant Der p 2-Fve fusion protein produced in rice suspension cell cultures has a great potential for allergy immunotherapy.     

Fed-batch cultivation of transgenic rice cells for the production of hCTLA4Ig using concentrated amino acids

RAmy3D promoter is capable of expressing high levels of recombinant proteins in response to the depletion of sugar in transgenic rice cell suspension cultures. For this reason, it is necessary to change the growth medium into sugar-free production medium to produce the target protein, human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig), using the inducible RAmy3D promoter. Since the two-stage culture is a complex process to perform in large-scale, a fed-batch method was evaluated with the addition of concentrated amino acids before the depletion of sugar to induce hCTLA4Ig production. This fed-batch culture was found to be effective and the production of hCTLA4Ig was enhanced up to 1.2-fold compared to that of two-stage cultures with medium exchange. In addition, when this fed-batch culture was performed in a 15-l stirred-tank bioreactor, maximum hCTLA4Ig level was 76.5 mg l⁻¹ at day 10.     

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 68.2 mg/L on day 5 in the culture medium with 40 mM 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 40 mM fumaric acid. Among the used combinations, the combination of 1% sucrose and 40 mM fumaric acid resulted in a yield of up to 53 mg/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 40 mM fumaric acid supplying-harvesting cycles was developed in flask scale culture. Recombinant bovine trypsin could be stably produced with a yield of up to 53–39 mg/L per cycle during five recycling cycles.     

Production of human caseinomacropeptide in recombinant Saccharomyces cerevisiae and Pichia pastoris

Caseinomacropeptide is a polypeptide of 64 amino acid residues (106–169) derived from the C-terminal part of the mammalian milk k-casein. This macropeptide has various biological activities and is used as a functional food ingredient as well as a pharmaceutical compound. The gene encoding the human caseinomacropeptide (hCMP) was synthesized and expressed with an α-factor secretion signal in the two yeast strains, Saccharomyces cerevisiae and Pichia pastoris. The complete polypeptide of the recombinant hCMP was produced and secreted in a culture medium by both the strains, but the highest production was observed in S. cerevisiae with a galactose-inducible promoter. In a fed-batch bioreactor culture, 2.5 g/l of the recombinant hCMP was obtained from the S. cerevisiae at 97 h.     

Constitutive expression of barley α-amylase in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by high-density cell culture

α-amy gene amplified from barley genome was cloned into MCS of pGAP9K to generate pGAP9K-α-amy which was then transformed into Pichia pastoris GS115 by electroporation. Transformants with multi-copies and high expression for the foreign gene were selected on G418 containing plate and expression analysis. The fermentation was carried out in a 50 l bioreactor with 20 l working volume, using a high-density cell culture method by continuously feeding with 50% glycerol-0.8% PTM4 to the growing culture for 54 h at 30°C. Under the control of GAP promoter (pGAP), α-amy gene was constitutively expressed. At the end of the fermentation, the α-AMY expression reached 125 mg/l, while the biomass growth was 186 as measured by absorption of 600 nm. The secreted α-AMY was purified to 97.5% by SP-Sepharose FF ion-exchange chromatography and affinity purification. The recombinant α-AMY showed activity on hydrolysis of starch.     

The modified rice αAmy8 promoter confers high-level foreign gene expression in a novel hypoxia-inducible expression system in transgenic rice seedlings

Expression of α-amylase genes in rice is induced not only by sugar starvation and gibberellin (GA) but also by O₂ deficiency. Promoters of two rice α-amylase genes, αAmy3 and αAmy8, have been shown to direct high-level production of recombinant proteins in rice suspension cells and germinated seeds. In the present study, we modified the cis-acting DNA elements within the sugar/GA response complex (SRC/GARC) of αAmy8 promoter. We found that addition of a G box and duplicated TA box leads to high-level expression of αAmy8 SRC/GARC and significantly enhances αAmy8 promoter activity in transformed rice cells and germinated transgenic rice seeds. We also show that these modifications have drastically increased the activity of αAmy8 promoter in rice seedlings under hypoxia. Our results reveal that the G box and duplicated TA box may play important roles in stimulating promoter activity in response to hypoxia in rice. The modified αAmy8 promoter was used to produce the recombinant human epidermal growth factor (hEGF) in rice cells and hypoxic seedlings. We found that the bioactive recombinant hEGF are stably produced and yields are up to 1.8 % of total soluble protein (TSP) in transformed rice cells. The expression level of synthetic hEGF containing preferred rice codon usage comprises up to 7.8 % of TSP in hypoxic transgenic seedlings. Our studies reveal that the modified αAmy8 promoter can be applicable in establishing a novel expression system for the high-level production of foreign proteins in transgenic rice cells and seedlings under hypoxia.     

Effects of culture media on hCTLA4Ig production and protein expression patterns in transgenic rice cell suspension cultures

The effects of culture media on the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) and intracellular protein expression patterns were investigated in transgenic rice cell suspension cultures. Using comparative proteomic analysis, changes in the intracellular proteome in different culture media were identified. Culture media were found to be an important factor for the production of the recombinant target protein in this expression system, which was under the control of the rice α-amylase 3D (RAmy3D) promoter. In terms of hCTLA4Ig production, the N6 medium produced a 3.7-fold higher level of protein than the AA medium. In addition, the N6 medium provided better protein stability and cell viability. In the intracellular proteome analysis, we identified eight proteomes that were differentially expressed. These results could provide valuable information for the improvement of cell growth and target protein production.     

Expression of bioactive human interferon-gamma in transgenic rice cell suspension cultures

We investigated the possibility of producing the therapeutic recombinant cytokine, Interferon-gamma (IFN-), in transgenic rice cell (Oryza sativa, cultivar TNG67) suspension cultures. We tested expression of two vector constructs, each harboring an Amy3 leader peptide and a C-terminus His 6 tag fused to a human IFN- cDNA, one driven by a sucrose-starvation inducible promoter (rice Amy3 promoter) and the other by a constitutive maize ubiquitin promoter, in rice cell suspensions, introduced via Agrobacterium tumefaciens. There was a significant difference in the amounts of recombinant IFN- protein produced by the Ups and Amy cell lines, as cytosolic and secretory proteins respectively. Immunological analysis of IFN- recombinant protein conferred a dose-dependent anti-dengue virus activity in human A549 cells, similar to the commercial product. We discuss the attractive attributes of using rice cell suspension system for the expression of therapeutic recombinant IFN-.     

Production of bioactive human granulocyte-colony stimulating factor in transgenic rice cell suspension cultures

Human granulocyte-colony stimulating factor (hG-CSF), a human cytokine, was expressed in transgenic rice cell suspension culture. The hG-CSF gene was cloned into the rice expression vector containing the promoter, signal peptide, and terminator derived from a rice alpha-amylase gene Amy3D. Using particle bombardment-mediated transformation, hG-CSF gene was introduced into the calli of rice (Oryza sativa) cultivar Dong-jin. Expression of the hG-CSF gene was confirmed by ELISA and Northern blot analysis. The amount of recombinant hG-CSF accumulated in culture medium from transgenic rice cell suspension culture on the sugar starvation was determined by time series ELISA. Biological activity of the plant derived hG-CSF was confirmed by measuring the proliferation of the AML-193 cells, and was similar to that of the commercial Escherichia coli-derived hG-CSF. In this paper, we discuss the attractive attributes of using rice cell suspension system for the expression of therapeutic recombinant hG-CSF.     

Stable expression of recombinant human coagulation factor XIII in protein-free suspension culture of Chinese hamster ovary cells

The recombinant a and bsubunits for human coagulation factor XIII were transfected into Chinese hamster ovary (CHO) cells. CHO cells were amplified and selected with methotrexate in adherent cultures containing serum, and CHO 1-62 cells were later selected in protein-free medium. To develop a recombinant factor XIII production process in a suspension culture, we have investigated the growth characteristics of CHO cells and the maintenance of factor XIII expression in the culture medium. Suspension adaptation of CHO cells was performed in protein-free medium, GC-CHO-PI, by two methods, such as serum weaning and direct switching from serum containing media to protein-free media. Although the growth of CHO cells in suspension culture was affected initially by serum depletion, cell specific productivity of factor XIII showed only minor changes by the direct switching to protein-free medium during a suspension culture. As for the long-term stability of factor XIII, CHO 1-62 cells showed a stable expression of factor XIII in protein-free condition for 1000 h. These results indicate that the CHO 1-62cells can be adapted to express recombinant human factor XIII in a stable maimer in suspension culture using a protein-free medium. Our results demonstrate that enhanced cell growth in a continuous manner is achievable for factor XIII production in a protein-free medium when a perfusion bioreactor culture system with a spin filter is employed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Optimization of the culture medium for recombinant protein production under the control of the <Emphasis Type="Italic">αAmy3</Emphasis> promoter in a rice suspension-cultured cell expression system

Many recombinant proteins have been produced successfully in a rice suspension-cultured expression system based on the αAmy3 promoter and signal peptide. Here, a transgenic rice cell line, H11, which can produce recombinant human serum albumin (rHSA) under conditions of sugar starvation, was cultured in various common rice-cell media, including MS, AA, and N6D, to determine the optimal medium for the production of rHSA in rice suspension-cultured cells. The yields of rHSA secreted into the N6D medium were 23.2 mg/L at an initial cell density of 3% and 49.6 mg/L at an initial cell density of 24%, which was 1.8-fold and twofold higher, respectively, than the yields obtained in MS. Moreover, the yield of rHSA was also twofold higher in N6D than in AA medium. Thus, rice suspension cells cultured in N6D medium had the highest rHSA productivity compared with MS and AA media. Proline is known to play roles in plant tolerance to stress. Consequently, we examined the effect of proline in N6D on rHSA production. A twofold increase in the concentration of proline in N6D increased the yield of rHSA to 32.3 mg/L at an initial cell density of 3% after 16 days, an increase of 39.2% compared with standard N6D. A beneficial effect of proline was also demonstrated on the production of recombinant mouse granulocyte-macrophage colony-stimulating factor from the G10 transgenic cell line. Our study showed that the supplementation of N6D with additional proline can promote the production of recombinant proteins in the αAmy3 promoter/signal peptide-based rice suspension-cultured system.     

Construction of an expression system for the secretory production of recombinant α-agarase in yeast

α-Agarase hydrolyzes the α-1,3 linkage of agarose yielding agaro-oligosaccharides. It is less well characterized than β-agarase. AgaA gene (2.3 kb ORF), encoding the α-agarase from Thalassomonas JAMB A33, was subcloned into both a constitutive and an inducible expression vector. Both the constructed plasmids, pVT-AgaA (ADH1 promoter) and pYInu-AgaA (GAL10 promoter), were transformed into Saccharomyces cerevisiae SEY2102 and FY833 and pPIC9-AgaA harboring the AOX1 promoter was transformed into Pichia pastoris GS115. The recombinant α-agarases were over-expressed with activities from 0.3 to 1.6 unit/ml, the highest being in the SEY2102/pYInu-AgaA transformant. Most of the recombinant α-agarase was extracellular because each plasmid possesses a signal sequence for the secretory production of α-agarase. In contrast, the Pichia host-vector expression system was unsuitable for the production of recombinant α-agarase. This is the first report of recombinant production of α-agarase in yeast for industrial use.     

Delayed supplementation of glycine enhances extracellular secretion of the recombinant α-cyclodextrin glycosyltransferase in Escherichia coli

The targeting of recombinant proteins for secretion to the culture medium of Escherichia coli presents significant advantages over cytoplasmic or periplasmic expression. However, a major barrier is inadequate secretion across two cell membranes. In the present study, we attempted to circumvent this secretion problem of the recombinant α-cyclodextrin glycosyltransferase (α-CGTase) from Paenibacillus macerans strain JFB05-01. It was found that glycine could promote extracellular secretion of the recombinant α-CGTase for which one potential mechanism might be the increase in membrane permeability. However, further analysis indicated that glycine supplementation resulted in impaired cell growth, which adversely affected overall recombinant protein production. Significantly, delayed supplementation of glycine could control cell growth impairment exerted by glycine. As a result, if the supplementation of 1% glycine was optimally carried out at the middle of the exponential growth phase, the α-CGTase activity in the culture medium reached 28.5 U/ml at 44 h of culture, which was 11-fold higher than that of the culture in regular terrific broth medium and 1.2-fold higher than that of the culture supplemented with 1% glycine at the beginning of culture.     

Salicylic acid inhibits gibberellin-induced alpha-amylase expression and seed germination via a pathway involving an abscisic-acid-inducible <Emphasis Type="Italic">WRKY</Emphasis> gene

It is well known that abscisic acid (ABA) antagonizes gibberellin (GA)-promoted seed germination. Recent circumstantial evidence suggests that salicylic acid (SA) also inhibits seed germination in maize and Arabidopsis. Our study shows that SA blocks barley seed germination in a dosage dependent manner. As an initial effort to addressing the mechanism controlling the crosstalk of SA, GA and ABA signaling in barley, we studied the regulation of α-amylases by SA and a WRKY gene whose expression is modulated by these hormones. Assays of α-amylase activity reveal that GA-induced α-amylase production in aleurone cells is inhibited by bioactive SA, but not its analogs, 3-hydroxybenzoic acid and 4-hydroxybenzoic acid. This inhibitory effect is unlikely due to repressing α-amylase secretion or inhibiting α-amylase enzyme activities. Northern blot analyses indicate that SA suppresses GA-induced expression of a barley low pI α-amylase gene (Amy32b). Because our previous data indicate that ABA-inducible and GA-suppressible WRKY genes inhibit the expression of α-amylase genes in rice, we studied the steady state mRNA levels of a barley WRKY gene, HvWRKY38. The expression of HvWRKY38 in barley aleurone cells is down-regulated by GA, but up-regulated by SA and ABA. However, the regulation of HvWRKY38 by SA appears to be different from that of ABA in term of the kinetics and levels of induction. Over-expression of HvWRKY38 in aleurone cells by particle bombardment blocks GA induction of the Amy32b promoter reporter construct (Amy32b-GUS). Therefore, HvWRKY38 might serve as a converging node of SA and ABA signal pathways involved in suppressing GA-induced seed germination. Zhen Xie and Zhong-Lin Zhang contributed equally to this work.     

The induction of α-amylase activity by sucrose starvation in suspension-cultured rice cells is regulated by polyamines

When suspension-cultured rice ( Oryza sativa L. cv. Tainan 5) cells were deprived of sucrose, α-amylase (EC 3.2.1.1) activity in the cells and the culture medium increased markedly. The increase in activity of α-amylase caused by sucrose starvation in the cells and the medium was strongly reduced in the presence of exogenously added spermine. Putrescine and spermidine also inhibited, though only slightly, the increase in α-amylase activity caused by sucrose starvation. Preincubation of the enzyme extract or enzyme in the medium with polyamines had no effect on α-amylase activity. Sucrose starvation resulted in lower polyamine levels in rice suspension cells. D-Arginine and α-methylomithine, inhibitors of polyamine biosynthesis, caused reduced levels of polyamines and increased activity of α-amylase in rice suspension cells cultured in the presence of sucrose. Our results indicate that the induction of α-amylase activity by sucrose starvation in rice suspension cells is mediated, at least partly, through the internal level of polyamines.     

Rosmarinic acid production by Lavandula vera MM cell-suspension culture

The time courses of growth and rosmarinic acid production by Lavandula vera MM cell suspension were investigated. The uptake of the main nutrients (sucrose, nitrogen, phosphorus, K, Ca, Mg) was followed during cultivation and the data on the physiology of the L. vera MM cell culture are presented. It was established that the cell culture synthesizes rosmarinic acid during the linear phase of growth for a relatively short period (between the 4th and 8th days of cultivation). The influence of sucrose concentration in the nutrient medium on cell growth and accumulation of rosmarinic acid by L. vera MM cell culture was investigated. The results showed that 7% sucrose in the nutrient medium ensured a steady growth of the cell suspension and increased the yield of rosmarinic acid (29.2 g/l dry biomass and 507.5 mg/l rosmarinic acid compared to 13.0 g/l dry biomass and 68.6 mg/l rosmarinic acid for the control cultivation with 3% sucrose). Received: 17 September 1996 / Received revision: 31 January 1997 / Accepted: 1 February 1997