• KSBB Journal
• /
• 제30권3호
• /
• pp.103-108
• /
• 2015

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.

• KSBB Journal
• /
• 제22권4호
• /
• pp.185-190
• /
• 2007

Productivity of secreted recombinant protein depends largely on its stability in the extracellular environment with protease. Most hGM-CSF produced by transgenic tobacco cell cultures and secreted to the medium was confirmed to be rapidly degraded by protease in medium. To increase the productivity, therefore, various protein stabilizers such as gelling agents such as carrageenan and alginate, polymers, polyols, and amino acids have been tested. The stability of hGM-CSF in spent medium without cells was improved by the presence of gelling agents. However, the reason for the enhanced production by the addition of gelling agents may be due to the increased expression level and permeability rather than stability. The addition of DMSO inhibited the cell growth, but improved specific yield. The others were not effective for stability as well as hGM-CSF production.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2003년도 생물공학의 동향(XIII)
• /
• pp.329-333
• /
• 2003

본 연구에서는 형질전환된 N. tabacum 배양에 있어서 배양 중반 저온으로의 변환이 세포에 미치는 영향과 hGM-CSF의 생산성 변화를 관찰하였다. 배양 중반 저온으로의 변환은 DCW의 증가와 세포크기의 감소를 보였다. Ascorbic acid의 첨가는 배양초기 세포 생존율의 감소를 완화시켰으며, 배양 중반 저온으로의 변환은 약간의 세포생존율 감소를 보였다. 저온으로의 변환, 저온 배양에서의 betaine 첨가, ascorbic acid 첨가 모두 배양 후반 세포 lysis 억제에 효과가 있었다. 배양 중반 저온으로의 변환시 배지내 단백질 분해 효소의 활성을 측정한 결과, 대조구 세포에 비해 낮은 단백질 분해 효소 활성을 나타내었다. 그로인해 배양 중반 이후 단백질 분해 효소에 의한 급격한 hGM-CSF 분해를 감소시킴으로써 상대적으로 대조구 세포에 비해 높은 hGM-CSF 생산성을 유지시켰다. Ascorbic acid를 첨가한 후 배양 도중 betaine(1 mM)을 첨가하여 저온으로 온도를 변환시, hGM-CSF의 생산성 대조구 세포에 비하여 최대 2.1배 까지 높게 유지시켰다.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2001년도 추계학술발표대회
• /
• pp.95-98
• /
• 2001

The effect of stabilizing polymer on hGM-CSF production was investigated in suspension cell cultures of transgenic tobacco. Secreted human GM -CSF from cell suspension cultures was detected in the medium at a maximum concentration of 180 ${\mu}g/L$ by ELISA. However, the secreted hGM -CSF was unstable in the medium, and rapidly degraded after day 5. In order to stabilize the secreted hGM-CSF, three stabilizing polymers were tested, polyethylene glycol, polyvinylpyrrolidone and gelatin. Gelatin was the most effective in stabilizing the secreted GM-CSF. Following the addition of 5% (w/v) gelatin, the maximum GM -CSF concentration reached 783 ${\mu}g/L$, a 4.6-fold increase over control.

• Journal of Applied Biological Chemistry
• /
• 제43권2호
• /
• pp.69-73
• /
• 2000

In order to understand the biological role of calmodulin in plants, transgenic plants expressing a mutant calmodulin (VU-4, Iys to ile-115) have been analyzed. We found that tobacco plants expressing VU-4 calmodulin have approximately twofold higher $\gamma$-aminobutyric acid (GABA) levels than the control plants. Cell suspension cultures established from the stem explants of the transgenic tobacco seedlings also have higher levels of GABA than the control cell cultures. Specific activity of glutamate decarboxylase (GAD), which catalyzes the decarboxylation of glutamate to $CO_2$ and GABA, of the transgenic tobacco cell extracts was about twofold higher than the activity of the control cell extracts. Western-blot analysis showed that the GAD is highly expressed in the transgenic tobacco plants. GAD partially purified from tobacco cell extracts showed approximately threefold $Ca^{2+}$/calmodulin-dependent activation. These data suggest that GABA synthesis in the transgenic tobacco plants is elevated, possibly due to higher levels of the calmodulin-dependent GAD enzyme and/or as a result of enhanced activation due to increased levels of the foreign calmodulin.

• KSBB Journal
• /
• 제30권2호
• /
• pp.82-90
• /
• 2015

Plant cell immobilization can protect plant cells from shear forces and increase the stability of gene. An additional advantage of immobilization is the easiness for performing continuous culture with cell recycling. Therefore plant cell immobilization can overcome the limitations of plant cell applications. In addition, target protein should be selected from pharmaceutical proteins to get rid of low expression level problem. The enhanced production of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was investigated in immobilized Nicotiana tabacum suspension cell cultures. When the cells were immobilized in polyurethane foam, specific production of hGM-CSF was higher than that in alginate bead immobilization. Optimum continuous culture condition was the addition of 60 g/L sucrose in growth media with exchanging media every 6 day. Under the same condition, specific hGM-CSF production was 7 times higher in a 500-mL spinner flask than that in 100-mL Erlenmeyer flasks. Therefore, development of an effective immobilization process would be possible when the advantage of easy cell recycling was used. Consequently, enhanced production of target proteins could be possible in immobilized continuous cultures when the advantages of immobilization were applied.

• Journal of Microbiology and Biotechnology
• /
• 제19권8호
• /
• pp.781-786
• /
• 2009

Small interfering synthetic double-stranded RNA (siRNA) was applied to suppress the expression of the human cytotoxic-T-Iymphocyte antigen 4-immunoglobulin (hCTLA4Ig) gene transformed in transgenic rice cell cultures. The sequence of the 21-nucleotide siRNA was deliberately designed and synthesized with overhangs to inactivate the expression of hCTLA4Ig. The chemically synthesized siRNA duplex was combined with polyethyleneimine (PEl) at a mass ratio of 1:10 (0.33 ${\mu}g$ siRNA:3.3 ${\mu}g$ PEl) to produce complexes. The siRNA complexes (siRNA+PEI) were labeled with Cy3 in order to subsequently confirm the delivery by fluorescent microscopy. In addition, the cells were treated with sonoporation at 40 kHz and 419W for 90 s to improve the delivery. The siRNA complexes alone inhibited the expression of hCTLA4Ig to 45% compared with control. The siRNA complexes delivered with sonoporation downregulated the production of hCTLA4Ig to 73%. Therefore, we concluded that the delivery of siRNA complexes into plant cells could be enhanced successfully by sonoporation.

• Plant Biotechnology Reports
• /
• 제5권3호
• /
• pp.245-254
• /
• 2011

This study describes an efficient protocol for Agrobacterium tumefaciens-mediated transformation of two subgroups of genotype AAA bananas (Musa acuminata cv. Pei Chiao and Musa acuminata cv. Gros Michel). Instead of using suspension cells, cauliflower-like bud clumps, also known as multiple bud clumps (MBC), were induced from sucker buds on MS medium containing $N^6$-Benzylaminopurine (BA), Thidiazuron (TDZ), and Paclobutrazol (PP333). Bud slices were co-cultivated with A. tumefaciens C58C1 or EHA105 that carry a plasmid containing Arabidopsis root-type ferredoxin gene (Atfd3) and a plant ferredoxin-like protein (pflp) gene, respectively. These two strains showed differences in transformation efficiency. The EHA105 strain was more sensitive in Pei Chiao, 51.3% bud slices were pflp-transformed, and 12.6% slices were Atfd3-transformed. Gros Michel was susceptible to C58C1 and the transformation efficiency is 4.4% for pflp and 13.1% for Atfd3. Additionally, gene integration of the putative pflp was confirmed by Southern blot. Resulting from the pathogen inoculation assay, we found that the pflp transgenic banana exhibited resistance to Fusarium oxysporum f. sp. cubense tropical race 4. This protocol is highly advantageous to banana cultivars that have difficulties in setting up suspension cultures for the purpose of quality improvement through genetic transformation. In addition, this protocol would save at least 6 months in obtaining explants for transformation and reduce labor for weekly subculture in embryogenic cell suspension culture systems.

• KSBB Journal
• /
• 제16권6호
• /
• pp.568-572
• /
• 2001

빛은 식물에서 성장과 발달을 비롯한 다양한 생리화학적인 역할은 지닌다. 본 연구는 hGM-CSF 유전자가 도입된 형질전한 담배의 callus를 현탁배양하여 hGM-CSF를 생산할 때에 빛을 조사하는 시간에 따른 hGM-CSF의 생산에 미치는 영향을 확인하고자 실시하였다. 24시간 명배양, 18시간 명배양과 암배양을 실시하여 세포성장과 분비된 총단백질, hGM-CSF 생산량을 비교 관찰하였다. 세포의 성장은 24시간 명배양일 때 건조중량이 14.4 g/L로 가장 높았다. 분비된 총단백질의 양은 세가지 경우에서 큰 차이를 관찰할 수가 없었지만, 단위 세포당 분비된 총단백질의 양은 암배양이 다른 것에 비해 1.5배 가량 높았다. hGM-CSF의 생산은 18시간 명배양 조건이 가장 좋았으며 최대생산량이 495.5ug/L에 이르렀다. 또한 분비된 총단백질에서 hGM-CSF가 차지하는 비율은 18시간 명배양이 24시간 명배양에 비해 최대 1.8배 높았다.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제8권3호
• /
• pp.187-191
• /
• 2003

Proteolytic enzymes existing in plant cell cultured media are the major reason for the loss of secreted human granulocyte-macrophage colony-stimulating factor (hGM-CSF). The addition of pepstatin, aprotinin and PMSF relatively decreased the proteolytic degradation of hGM-CSF in a conditioned medium, but sufficient prevention against the proteolytic activity could not be obtained with chemical protease inhibitors. Gelatin, as a competitive substrate for protease, showed a stabilizing effect in a conditioned medium. Compared to the initial hGM-CSF concentration in a conditioned medium. with 10 g/L of gelatin, 68% of the hGM-CSF remained after 5 days. In a cell culture experiment, 5 g/L of gelatin significantly stimulated the hGM-CSF production and accumulation in culture media, with no growth inhibition. compared to the controls (4.72 $\mu\textrm{g}$/L), the extracellular hGM-CSF level could be increased to 39.78 $\mu\textrm{g}$/L with the addition of 5 g/L of gelatin.