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.

Immobilization of transgenic plant cells towards bioprinting for production of a recombinant biodefense agent.

Transgenic rice cells (Oryza sativa) producing recombinant butyrylcholinesterase (BChE) as a prophylactic/therapeutic against organophosphate nerve agent poisoning, cocaine toxicity, and neurodegenerative diseases like Alzheimer's were immobilized in a polyethylene glycol-based hydrogel. The cells were sustained for 14 days in the semi-solid matrix, undergoing a growth phase from days 0-6, a BChE production phase in sugar-free medium from days 6-12, and a growth/recovery phase from days 12-14. Throughout this period, the cells maintained similar viability to those in suspension cultures and displayed analogous sugar consumption trends. The rice cells in the hydrogel also produced a significant amount of active BChE, comparable to the levels produced in liquid cultures. A considerable fraction of this BChE was secreted into the media, allowing for easier product separation. To the best of our knowledge, this proof-of-concept is the first report of immobilization of recombinant plant cells for continuous production of high-value heterologous proteins. This work serves as a foundation for further investigation towards plant cell bioprinting and the development of a simple, efficient, robust, modular, and potentially field-deployable bioreactor system for the manufacture of biologics.

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.

Local supplementation with plant-derived recombinant human FGF2 protein enhances bone formation in critical-sized calvarial defects.

Numerous studies have demonstrated the advantages of plant cell suspension culture systems in producing bioactive recombinant human growth factors. This study investigated the biological activity of recombinant basic human fibroblast growth factor (rhFGF2) protein produced by a plant culture system to enhance new bone formation in a bone defect mouse model. The human FGF2 cDNA gene was cloned into a plant expression vector driven by the rice α-amylase 3D promoter. The vector was introduced into rice calli (Oryza sativa L. cv. Dongjin), and the clone with the highest expression of rhFGF2 was selected. Maximum accumulation of rhFGF2 protein (approximately 28 mg/l) was reached at 13 day post-incubation. Male C57BL/6 mice underwent calvarial defect surgery and the defects were loaded with absorbable collagen sponge (ACS) only (ACS group) or ACS impregnated with 5 µg of plant-derived rhFGF2 (p-FGF2) protein or E. coli-derived rhFGF2 (e-FGF2) protein. Similar to the effects of e-FGF2, local delivery with p-FGF2 enhanced bone healing in the damaged region to higher levels than the ACS group. Exogenous addition of p-FGF2 or e-FGF2 exhibited similar effects on proliferation, mineralization, and osteogenic marker expression in MC3T3-E1 cells. Together, the current findings support the usefulness of this plant-based expression system for the production of biologically active rhFGF2.

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.

Production and Purification of Recombinant Glucocerebrosidase in Transgenic Rice Cell Suspension Cultures.

Gaucher disease, which is caused by deficiency of glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy. Plant-based systems produce glycoproteins and can be combined with targeting strategies to generate proteins with terminal mannose structures for macrophage uptake. However, the gliding step for the purification is essential since the produced protein still exists inside cells. In the case of rice-amylase 1A (RAmy1A) secretion signal peptide, GCD protein is secreted outside of cells and simplifies the purification step. Here, an established cell line was confirmed as having fundamental characteristics of growth and production. GCD from transgenic calli was examined by Western blot analysis and compared with that from Chinese hamster ovary (CHO) cells. Calli expressing high levels of GCD were used to establish suspension cell lines. Growth and production characteristics were investigated in suspension cell cultures. Production of GCD in suspension cultures was confirmed upon induction for 12-24 h. The amount of GCD in medium increased until 60-84 h and decreased thereafter. Purification of GCD was performed in three steps (ion exchange, hydrophobic interaction, and size exclusion chromatography) and verified. Purified GCD was able to hydrolyze the synthetic substrate. Thus, a rice expression system could be a suitable alternative to GCD expression in mammalian cells.

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.

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.

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.

Bioreactor engineering using disposable technology for enhanced production of hCTLA4Ig in transgenic rice cell cultures.

Two kinds of disposable bioreactors, air-lift disposable bioreactors (ADB) and wave disposable bioreactors (WDB) were compared with stirred-tank reactors (5-L STR). These bioreactors were successfully applied to transgenic rice cell cultures for the production of recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig). In both systems, a fed-batch culture method was used to produce hCTLA4Ig efficiently by feeding concentrated amino acids and production levels were enhanced when dissolved oxygen (DO) level was regulated at 30% using pure oxygen sparging. Agitation and aeration rate during cultivation in ADB and WDB were determined by the same mixing time. The results in both disposable bioreactors showed similar values in maximum cell density (11.9 gDCW/L and 12.6 gDCW/L), doubling time (4.8- and 5.0-day), and maximum hCTLA4Ig concentration (43.7 and 43.3 mg/L). Relatively higher cell viability was sustained in the ADB whereas hCTLA4Ig productivity was 1.2-fold higher than that in WDB. The productivity was improved by increasing aeration rate (0.2 vvm). Overall, our experiments demonstrate pneumatically driven disposable bioreactors are applicable for the production of recombinant proteins in plant cell cultures. These results will be useful for development and scale-up studies of disposable bioreactor systems for transgenic plant cell cultures.