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

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

• KSBB Journal
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• 제31권4호
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• pp.284-290
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• 2016

In this research, recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was produced by transgenic rice cells. RAmy3D promoter was used for overcome the limitation of low expression level in transgenic plant cells, and the secretion of target protein was accomplished by signal peptide. However, the RAmy3D promoter system which can be induced only by sugar starvation causes the decrease of cell viability. As a result, cell death promotes the release of protease which degrades the target proteins. The protein stability and productivity can be significantly influenced by proteolysis activity. Therefore, development of new strategies are necessary for the in situ recovery of target proteins from cell culture media. In this study, in situ recovery was performed by various strategies. Direct addition of Protein A resin with nylon bag leads to cell death by increased shear stress and decrease in production of hCTLA4Ig by protease. Medium exchange through modified flask could recover hCTLA4Ig with high cell viability and low protease activity, on the other hand, the productivity was lower than that of control. When in situ recovery was conducted at day 7 after induction in air-lift bioreactor, 1.94-fold of hCTLA4Ig could be recovered compared to control culture without in situ recovery. Consequently, in situ recovery of hCTLA4Ig from transgenic rice cell culture could enhance productivity significantly and prevent degradation of target proteins effectively.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.186-186
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• 2005

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권1호
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• pp.12-16
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• 2004

Partitioning of human granulocyte-macrophage colony stimulating factor (hGM-CSF) was achieved in the aqueous two-phase systems (ATPSs) using a crude extract of transgenic tobacco cell suspension culture. This study examined the effects of polyethylene glycol (PEG) molecular weight and concentration and the effects of sodium phosphate concentration in different PEG/sodium phosphate systems on the partition coefficient, K. The best ATPS system was 5% PEG 8,000/1.6 M sodium phosphate after 2 h of incubation at room temperature. In this system, hGM-CSF was partitioned in the PEG-rich phase with a yield of 57.99% and K$\_$hGM-CSF/ of 8.12. In another system, 3% PEG 10,000/1.6 M sodium phosphate, hGM-CSF was also partitioned primarily in the top phase with a yield of 45.66% and K$\_$hGM-CSF/ of 7.64 after 2 h of incubation at room temperature.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 춘계학술발표대회
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• pp.317-320
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• 2000

유전자 재조합된 Nicotiana tabacum을 이용하여 동결 보존 후의 세포 생장을 관찰하였다. 형질 전환된 Nicotiana tabacum의 동결 보존에서 회복배지에 계면 활성제와 산소 전달 물질을 첨가한 경우 첨가하지 않은 경우에 비해 세포 생장을 증대 시켰고 산소 전달 물질 보다 계면활성제의 영향이 크다는 결과를 관찰하였다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권5호
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• pp.442-448
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• 2006

Human lactoferrin (hLf) is an iron-binding glycoprotein that has been considered to play many biological roles in the human, including the stimulation of the immune system, antimicrobial and anti-inflammatory effects, and regulation of iron absorption. We generated transgenic Siberian ginseng (Acanthopanax senticosus) cell cultures producing a functional hLf protein using the signal peptide sequence from the endoplasmic reticulum and driven by an oxidative stress-inducible SWPA2 promoter which is highly expressed in plant cell cultures. The production of hLf increased proportionally to cell growth and showed a maximal level (up to 3.6% of total soluble protein) at the stationary phase in suspension cultures. Full-length hLf protein was identified by immunoblot analysis in transgenic cell cultures of Siberian ginseng. Recombinant hLf (rhLf) was purified from suspension cells of Siberian ginseng by ammonium sulfate precipitation, cation-exchange and gel filtration chromatography. N-terminal sequences of rhLf were identical to native hLf (nhLf). The overall monosaccharide composition of rhLf showed the presence of plant specific xylose while sialic acid is absent. Antibacterial activity of purified rhLf was higher than that of nhLf. Taken together, we anticipate that medicinal Siberian ginseng cultured cells, as demonstrated by this study, will be a biotechnologically useful source for commercial production of functional hLf not requiring further purification.

• 생명과학회지
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• 제14권2호
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• pp.368-373
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• 2004

광범위 제초제인 Bast $a^{(R)}$에 대해 저항성을 가지는 형질전환체 벼를 개발하였다. Bar유전자를 함유하고 있는 플라스미드 pCaMV35S::Bar를 embryogenic 현탁 배양체로부터 분리한 벼의 원형질체에 도입하였다. Phosphinotricin에 대해 저항성을 가지는 형질전환체 식물체들이 재분화되었고, 이들을 15 mg/l phosphinotricin이 함유한 배지에서 다시 선별하였다. 형질전환체 벼에서 bar유전자의 삽입과 발현을 Southern과 Northern blot분석으로 확인하였고, 또한 $R_1$ 형질전환 식물체들을 PAT 활성 assay로 재차 유전자 발현을 확인하였다. Bar 유전자는 다음 세대인 $R_1$ 식물체에서 3:1 멘델 유전 양상을 나타내었고, 형질전환체 $R_1$과 $R_2$ 식물체들은 fieild에서 살포되는 제초제 양만큼 Bast $a^{(R)}$ 를 살포했을 때 제초제 저항성을 나타내었다..

• KSBB Journal
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• 제18권6호
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• pp.435-439
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• 2003

형질 전환된 담배 seed에서 담배 식물체를 유도하여 White 액체 배지에서 기관 배양하였다. 암조건, sucrose 2%의 조건에서 좋은 growth pattern을 나타내었고, 계대 배양은 외마디법을 이용하여 2주마다 하였다. 기관 배양에서 hGM-CSF production pattern을 보면, intracellular에서는 큰 변화 없이 약 30 ng/g의 일정한 농도를 나타내었다. Extracellular에서 hGM-CSF 농도는 배양 6일 이후부터 급속하게 증가하기 시작하여 배양 12일째에 약 0.2ng/$m\ell$의 농도를 나타낸다. 기관배양은 다른 식물세포 배양 시스템에 비해 생산되어진 단백질의 안정성이 크다는 장점에 비해 세포 내에서 배지 내로의 단백질 분비가 적다는 단점이 있다. 이를 극복하기 위해 다양한 permeabilizing agents를 투여하여 담배 세포의 permeability를 증가시키고자 하였다. 그 결과, Pluronic F-68과 PEG8000을 첨가한 경우 담배 세포에서 배지 내로의 단백질 분비가 원활해졌음을 확인할 수 있었다.

• KSBB Journal
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• 제26권5호
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• pp.386-392
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• 2011

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig), an immunosuppressive agent, was expressed in transgenic rice cells using RAmy3D promoter and RAmy1A signal peptide for the inducible production and secretion into culture media by sugar depletion. In this study, sodium butyrate was used as a small molecular enhancer (SME) to enhance the production of hCTLA4Ig in transgenic rice cell suspension cultures. When 1 mM sodium butyrate was added in sugar-free media, relative viability was not reduced, while the productivity was improved 1.3-fold. In addition, by supplementing 87 mM sodium pyruvate as an alternative energy source during the production phase, death rate of the cells was decreased. When sodium pyruvate was not added, most cells became dead at day 6. However, by adding sodium pyruvate, 18% of viability can be maintained until day 10 and the production of hCTLA4Ig was enhanced 1.4-fold. When the combination of sodium pyruvate and sodium butyrate at optimum concentrations was added, the highest viability and hCTLA4Ig production could be obtained. The highest level of hCTLA4Ig reached up to 35 mg/L at day 10.