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Enzyme replacement therapy for lysosomal storage diseases usually requires recombinant enzymes containing mannose-6-phosphate (M6P) glycans for cellular uptake and lysosomal targeting. For the first time, a strategy is established here for the in vitro mannosyl-phosphorylation of high-mannose type N-glycans that utilizes a recombinant Mnn14 protein derived from Saccharomyces cerevisiae. Among a series of N-terminal- or C-terminal-deleted recombinant Mnn14 proteins expressed in Pichia pastoris, rMnn1477-935 with deletion of N-terminal 76 amino acids spanning the transmembrane domain (46 amino acids) and part of the stem region (30 amino acids), showed the highest level of mannosyl-phosphorylation activity. The optimum reaction conditions for rMnn1477-935 were determined through enzyme assays with a high-mannose type N-glycan (Man8GlcNAc2) as a substrate. In addition, rMnn1477-935 was shown to mannosyl-phosphorylate high-mannose type Nglycans (Man7-9GlcNAc2) on recombinant human lysosomal alpha-glucosidase (rhGAA) with remarkably high efficiency. Moreover, the majority of the resulting mannosyl-phosphorylated glycans were bis-form which can be converted to bis-phosphorylated M6P glycans having a superior lysosomal targeting capability. An in vitro N-glycan mannosyl-phosphorylation reaction using rMnn1477-935 will provide a flexible and straightforward method to increase the M6P glycan content for the generation of "Biobetter" therapeutic enzymes.

Although plant cell cultures produce low yields of recombinant proteins compared to other production systems, a dramatic increase of the heterologous protein production in transgenic rice cells was achieved with the alpha-amylase isozyme 3D (RAmy3D) promoter system. However, this expression system has inherent limitations in that gene expression is initiated by sucrose/glucose deprivation, concurrently triggering starvation-derived autophagy and rapid cell death. Decreased viability and culture longevity subsequently prevent further increment of production. In this study, we introduced autophagy inducers and inhibitors in the rrhGAA-producing transgenic rice cell cultures in order to explore their effects on production controlled by the RAmy3D promoter. The autophagy inducers rapamycin and CCI-779 increased autophagosome and autolysosome while concanamycin A1 and bafilomycin A successfully decreased autolysosome. Interestingly, autophagy inhibitors improved viability, DCW loss, and rrhGAA production, while autophagy inducers deteriorated these profiles compared to the control. As the production conditions under the death phase may facilitate protein degradation, and subsequently exacerbate functional activity, the size variant distribution and enzyme activity of the purified rrhGAAs were evaluated. However, no significant difference in rrhGAA degradation as well as GAA activity was observed compared to the control condition, thus indicating that the autophagy regulation is an efficient approach to increase protein yield in rice cell culture system for rrhGAA production.

Enzyme replacement therapy for lysosomal storage diseases usually requires recombinant enzymes containing mannose-6-phosphate (M6P) glycans for cellular uptake and lysosomal targeting. For the first time, a strategy is established here for the in vitro mannosyl-phosphorylation of high-mannose type

A reproducible method for cryopreservation of transgenic rice cells (Oryza sativa L. cv. Dongjin) producing recombinant human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (hCTLA4Ig) has been established. Here, we assessed recovery media and investigated recombinant protein homogeneity after long-term preservation. For recovery of cryopreserved transgenic rice cells, AA medium was suitable in terms of both morphology and production of hCTLA4Ig. There were no differences in cell growth, sugar consumption, and hCTLA4Ig production between non-cryopreserved and cryopreserved cells for up to 1 month. hCTLA4Ig produced from cryopreserved cells was identical that of hCTLA4Ig from non-cryopreserved cells, as determined by analysis of its molecular weight and isoforms. For long-term preservation, cell viability was stably maintained at 61% for 26 months. In conclusion, these results demonstrate the possibility for reproducible cryocell-banking of transgenic rice cells without changes in the characteristics of cells and target proteins.

Various polymers were used as transfection factors for small interfering RNA (siRNA) to effectively suppress human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) gene in transgenic rice cells. Five kinds of polymers (PEI, PVA, PVP, and 8 and 20 kDa PEGs) were applied for delivery of siRNA with lipofectamine used as a control. In the cytotoxicity test, all polymers except 8 kDa PEG showed nontoxicity in relation to cell viability. For transfection efficiency, polyplexes composed of siRNA and PEG (20 kDa) did not significantly reduce production of intracellular hCTLA4Ig. On the other hand, siRNA + PEI polyplexes showed the most effective suppression efficiency with regards to production of intracellular hCTLA4Ig among all other polyplexes (PVA, PVP, and PEG (8 kDa)). Effects of molecular weight ratios of siRNA:PEI were investigated to obtain optimal transfection efficiency and avoid excessive damage to cells. PEI-based polyplexes with a 1:10 ratio of siRNA:PEI reduced production of intracellular hCTLA4Ig up to 70.6% without alteration of cell viability. These results demonstrate that PEI-based polyplexes are easy to prepare, inexpensive, non-toxic, and effective to deliver siRNA to transgenic plant cell cultures.

In this system, rice cells were genetically modified to express human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) using RAmy3D promoter induced by sugar depletion. Even though the target protein fused with signal sequence peptide, plant cell wall can be a barrier against secretion of recombinant proteins. Therefore, hCTLA4Ig can be trapped inside cell wall or remained in intracellular space. In this study, to enhance the secretion of hCTLA4Ig from cytoplasm and cell walls into the medium, permeabilizing agents, such as dimethyl sulfoxide (DMSO), Triton X-100 and Tween 20, were applied in transgenic rice cell cultures. When 0.5% (v/v) of DMSO was added in sugar-free medium, intracellullar hCTLA4Ig was increased, on the other hand, the secreted extracellular hCTLA4Ig was lower than that of control. DMSO did not give permeable effects on transgenic rice cell cultures. And Triton X-100 was toxic to rice cells and also did not give enhancing permeability of cells. When 0.05% (v/v) Tween 20 was added in rice cell cultures, however, intracellular hCTLA-4Ig was lower than that of control cultures. And the maximum 44.76 mg/L hCTLA4Ig was produced for 10 days after induction, which was 1.4-fold increase compared to that of control cultures. Especially, Tween 20 at 0.05% (v/v) showed the positive effect on the secretion of hCTLA4Ig though the decrease of intracellular hCTLA4Ig. Also, Tween 20 as a non-toxic surfactant did not affect the cell growth, cell viability and protease activity. In conclusion, secretion of hCTLA4Ig could be increased by enhancing permeability of cells regardless of the cell growth, cell viability and protease activity.

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.

Gaucher disease is an inherited metabolic disease caused by genetic acid β -glucosidase (GBA) deficiency and is currently treated by enzyme replacement therapy. For uptake into macrophages, GBA needs to carry terminal mannose residues on their N-glycans. Knockout mutant rice of N-acetylglucosaminyltransferase-I (gnt1) have a disrupted N-glycan processing pathway and produce only glycoproteins with high mannose residues. In this study, we introduced a gene encoding recombinant human GBA into both wild-type rice (WT) and rice gnt1 calli. Target gene integration and mRNA expression were confirmed by genomic DNA PCR and Northern blotting, respectively. Secreted rhGBAs in culture media from cell lines originating from both WT (WT-GBA) and rice gnt1 (gnt1-GBA) were detected by Western blotting. Each rhGBA was purified by affinity and ion exchange chromatography. In vitro catalytic activity of purified rhGBA was comparable to commercial Chinese hamster ovary cell-derived rhGBA. N-glycans were isolated from WT-GBA and gnt1-GBA and analyzed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The amounts of high mannose-type N-glycans were highly elevated in gnt1-GBA (100%) compared to WT-GBA (1%).

Glycoengineering of plant expression systems is a prerequisite for the production of biopharmaceuticals that are compatible with animal-derived glycoproteins. Large amounts of high-mannose glycans such as Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 (Man7/8/9), which can be favorably modified by chemical conjugation of mannose-6-phosphate, are desirable for lysosomal enzyme targeting. This study proposed a rice cell-based glycoengineering strategy using two different mannosidase inhibitors, kifunensine (KIF) and swainsonine (SWA), to increase Man7/8/9 glycoforms of recombinant human acid α-glucosidase (rhGAA), which is a therapeutic enzyme for Pompe disease. Response surface methodology was used to investigate the effects of the mannosidase inhibitors and to evaluate the synergistic effect of glycoengineering on rhGAA. Both inhibitors suppressed formation of plant-specific complex and paucimannose type N-glycans. SWA increased hybrid type glycans while KIF significantly increased Man7/8/9. Interestingly, the combination of KIF and SWA more effectively enhanced synthesis of Man7/8/9, especially Man9, than KIF alone. These changes show that SWA in combination with KIF more efficiently inhibited ER α-mannosidase II, resulting in a synergistic effect on synthesis of Man7/8/9. In conclusion, combined KIF and SWA treatment in rice cell culture media can be an effective method for the production of rhGAA displaying dominantly Man7/8/9 glycoforms without genetic manipulation of glycosylation.

Production of foreign proteins by transgenic plant cell cultures has several advantages such as post-translational modification, low risk of product contamination and low-cost production and purification. However, target proteins are degraded by extracellular proteases existing in the media. A solution to this problem is the use of perfusion culture and ion exchange chromatography for the application of integrated bioprocess using in situ recovery. With this method, production of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was investigated in this study. First, optimization of cell concentration during the induction phase for the production of hGM-CSF was examined. As cell concentration increased, the level of hGM-CSF was decreased due to the presence of extracellular proteases. Induction using sugarfree media produced 33% more hGM-CSF. The effects of pH on the binding of hGM-CSF to cationic and anionic exchange resins were also investigated. In terms of stability, optimal pH was found to be 5~7. In the case of using buffer exchange when CM-Sepharose was used as a cationic exchange resin, optimal pH for binding was 4.8 and adsorption yield was 77%. When DEAE-Sepharose was used as an anionic exchange resin, it was 5.5 (74%). Without buffer exchange, optimal pH was 4.6 (84%). From these results, an integrated bioprocess using in situ recovery with simultaneous production and separation of foreign protein in transgenic plant cell suspension cultures was found to be feasible.

수분은 제품의 물리적 변화, 미생물 성장, 화학적 반응을 일으켜 품질을 저하시키는 주요 요인이 된다. 본 연구는 기능성 포장 필름으로 응용하기 위하여 수분을 흡착력이 우수한 폴리아크릴산 나트륨 염(polyacrylic acid partial sodium salt, PAPSS)을 선상저밀도폴리에틸렌(LLDPE) 고분자 수지에 함침된 수분 흡착 기능을 가진 복합 필름을 제조하였다. 제조한 수분 흡착 복합 필름은 PAPSS 물질 입자가 LLDPE 고분자 매트릭스에 잘 분포되어 있음을 확인하였다. 또한 PAPSS 첨가량이 증가됨에 따라 필름의 투명도가 저하되었다. 고분자에 첨가되는 PAPSS 물질의 증가는 복합 필름의 인장강도와 신장률 값이 감소됨을 확인할 수 있었으나 각각 0.5, 1, 그리고 2% PAPSS 필름들 간에는 큰 유의적 차이를 보이지 않았다. 반면 4% PAPSS 함침 필름은 다른 농도로 PAPSS 물질이 함침된 필름과 비교하여 인장강도 및 신장률 값의 급격한 감소를 나타냈다. PAPSS 함량의 증가는 필름의 산소 및 수분 투과도 값이 감소됨이 관찰되어 고분자에 PAPSS 첨가는 필름의 가스 차단의 개선을 나타냈다. PAPSS 함침 필름들은 상대적으로 높은 습도 환경 조건에서 우수한 수분 흡착율을 나타내는 것을 확인하였으며, 특히 4% PAPSS 함침 필름이 다른 PAPSS 농도 함침 필름들에 비하여 높은 수분 흡착율을 보였다. $25^{\circ}C$ 조건에서 필름 샘플의 수분 흡착 등온 곡선에 가장 적합하게 묘사되는 최적의 모델 방정식은 GAB 모델로 나타냈다. PAPSS 첨가는 고분자 필름의 결정화 및 녹는 온도 변화에 영향을 보였으나, 열중량 변화에는 큰 차이가 없이 안정된 열적 특성을 나타냈다. 【Moisture is a major factor causing the deteriorative physical change, microbial growth, and chemical reaction of the products. In this study, the moisture absorbing composite films have been prepared with moisture absorbing material of polyacrylic acid partial sodium salt (PAPSS) impregnated on LLDPE polymer for the functional packaging applications. The results showed that PAPSS impregnated film illustrated uniformly dispersed PAPSS particles in the LLDPE polymer matrix. The transparency of the PAPSS impregnated film decreased slightly at higher PAPSS concentrations. An increase in the PAPSS content for moisture-absorbing films showed a similar decrease in tensile strength, percent elongation at break, and tear strength. Their values of films impregnated with PAPSS of 0.5, 1, and 2% showed no significant difference. Meanwhile, 4% PAPSS films significantly decreased the values of mechanical properties compared to the films impregnated with different PAPSS levels. Values of the oxygen permeability and water vapor permeability for PAPSS impregnated films decreased significantly with greater PAPSS. The results indicate that 4% PAPSS impregnated in LLDPE films had high affinity of moisture absorbencies compared to the other films. The mathematical equation that best described the moisture sorption isotherm of each film sample was the GAB equation at $25^{\circ}C$ . The crystallization and melting temperatures of PAPSS films were influenced by the addition of PAPSS material, but showed good thermal stability.】

Moisture-absorbing film impregnated with synthesized attapulgite with acrylamide (ATPGAA) was developed. The effects of ATPGAA concentrations of 0.5%, 1%, 2%, and 4% (w/w) in a linear low-density polyethylene polymer (LLDPE) matrix on the film properties were determined by measuring the physical properties, the oxygen and water vapor barriers, and moisture absorbency. The 4% ATPGAA film had high moisture absorbing efficiency. A packaging system fabricated from ATPGAA-impregnated films was applied to a dried-seasoned laver to evaluate its change in quality during storage. Seasoned laver packaged with ATPGAA-impregnated films had significantly greater crispness in texture and a lower peroxide value during storage than that packed with LLDPE film. The surface color of the seasoned laver packaged by ATPGAA-impregnated films was more stable than the control film. This study shows that moisture-absorbing films could serve as a packaging system to improve the storage quality of moisture-sensitive products.

Most of the technical know-how and experience of bioreactor engineering is applicable to plant cell cultures. In this study, transgenic rice cell cultures using RAmy3D promoter were used for the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig). In process aspect, the rice cells during production phase are strongly influenced by hydrodynamic stresses, such as shear stress and bubble burst. Therefore, the effects of agitation and aeration rates on cell growth and hCTLA4Ig production were investigated in a 3-L multi-bioreactor. By increasing over 240 rpm, the detrimental effects on cell growth and hCTLA4Ig production were observed. At an aeration rate of 0.3 vvm, relative cell viability sharply decreased 2 days earlier than those of lower aeration rates. In addition, it was confirmed that the specific yields and the specific productivity at 0.3 vvm were superior to those values at 0.05 vvm. Overall, higher aeration rate showed the improved hCTLA4Ig production in combination experiment. High aeration rates in general, however, have an undesired effect as excessive aeration was found to negatively affect the quality of hCTLA4Ig. Consequently, the hydrodynamic conditions must be tightly regulated during bioreactor operation in order to enhance hCTLA4Ig productivity and quality in transgenic rice cell 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.

Rice (Oryza sativa) is one of the major food crops in world agriculture, especially in Asia. However, the possibility of subsequent occurrence of flood and drought is a major constraint to its production. Thus, the unique behavior of rice toward flooding and drought stresses has required special attention to understand its metabolic adaptations. However, despite several decades of research investigations, the cellular metabolism of rice remains largely unclear. In this study, in order to elucidate the physiological characteristics in response to such abiotic stresses, we reconstructed what is to our knowledge the first metabolic/regulatory network model of rice, representing two tissue types: germinating seeds and photorespiring leaves. The phenotypic behavior and metabolic states simulated by the model are highly consistent with our suspension culture experiments as well as previous reports. The in silico simulation results of seed-derived rice cells indicated (1) the characteristic metabolic utilization of glycolysis and ethanolic fermentation based on oxygen availability and (2) the efficient sucrose breakdown through sucrose synthase instead of invertase. Similarly, flux analysis on photorespiring leaf cells elucidated the crucial role of plastid-cytosol and mitochondrion-cytosol malate transporters in recycling the ammonia liberated during photorespiration and in exporting the excess redox cofactors, respectively. The model simulations also unraveled the essential role of mitochondrial respiration during drought stress. In the future, the combination of experimental and in silico analyses can serve as a promising approach to understand the complex metabolism of rice and potentially help in identifying engineering targets for improving its productivity as well as enabling stress tolerance.