• 생명과학회지
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• 제17권11호
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• pp.1497-1504
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• 2007

사람의 혈소판조절인자 (TPO)의 분비와 기능을 분석하기 위하여 사람 간 cDNA로부터 TPO cDNA를 분리하여 동물세포에서 재조합체를 생산하였다. 또한, 당쇄의 기능분석을 위하여 6개의 당쇄첨가부위를 Ala으로 치환하여 각각의 돌연변이체 재조합체도 생산하여 이들의 생리활성분석을 위하여 피하주사하여 혈소판의 증가여부를 분석하였으며, 체내 약동학검사를 위하여 꼬리 정맥에 재조합체를 주사하여 24시간까지 혈액을 채취하였다. Wild-type TPO는 효과적으로 분비하였으나, 크로닝에서 분석되어 진 116개 아미노산이 삭제된 돌연변이체는 배양상층으로 분비되지 않았다. N-linked 당쇄첨가 부위는 3번과 4번을 제외하고는 거의 비슷한 발현양상을 나타내었다. 특히 4번당쇄부위는 TPO의 분비에 중요한 역할을 하는 것으로 나타났다. 재조합체 10ng을 피하주사에 의하여 체내 혈소판이 유의적으로 증가하였으며, 5ng을 이용한 약동학 분석결과 1시간에 최대로 증가하였으며 그 이후 급격하게 감소하여 10시간에는 거의 존재하지 않았다. 따라서, 이러한 연구는 고 활성을 가지는 유전자 재조합체 TPO의 생산을 가능하게 하고, 또한 새로운 분자의 TPO를 가능하게 할 것으로 사료된다.

• Reproductive and Developmental Biology
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• 제32권1호
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• pp.45-49
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• 2008

We have previously produced transgenic (TG) mice expressing the human lactoferrin (hLF), interleukin-10 (hIL-10), and thrombopoietin (hTPO) proteins in the milk. In this study, we examined whether simple crossbreeding between two kids of a single transgenic mouse can produce double transgenics co-expressing two human proteins.. The hLF male, and the hIL-10 male were crossbred with the hIL-10 and hTPO females, and the hTPO female, respectively. PCR analysis for genotyping showed 32%, 23% and 24% double transgenic rates for hLF/hIL-10, hLF/hTPO, and hIL-10/hTPO transgenes, respectively. We analyzed the expression levels of the human proteins from double transgenic mice and compared those with their single transgenic siblings. All double transgenic co-expressed two human proteins at comparable levels to singles', unless hTPO was not co-expressed: for hLF, 1.1 mg/ml in hLF/hIL-10, whereas 0.5 mg/ml in hLF/hTPO; for hIL-10, 4.1 mg/ml in hIL-10/hLF, whereas 1.4 mg/ml in hIL-10/hTPO. Ihe downregulation of hTPO to half level of singles' was observed in double transgenic mice. The possible reason why hTPO co-expressed might lead to down-regulation of another human protein was discussed. These results suggested that double transgenic generated by crossbreeding between two singles' could be useful system for bioreactor.

• Molecules and Cells
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• 제19권2호
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• pp.198-204
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• 2005

Both erythropoietin (EPO) and the short-form thrombopoietin (TPO) were expressed at low levels whereas the long-form TPO was expressed at high levels in transgenic animals. To elucidate the role of carboxy-terminal half of the long-form TPO which is absent in the short-form, we generated recombinant TPO or EPO expression vectors which contain or lack the carboxy-terminal half of TPO and examined their expression in the HC11 and 293 cells. The long-form TPO was expressed higher than the short-form regardless of the cell types, transfection modes, and promoters. When 3'-half of the long-form TPO cDNA was placed downstream of the EPO cDNA to act as a 3'-untranslated region, expression of EPO was moderately increased at the RNA level, however, no remarkable increase was observed at the protein level. These results suggest that the low expression of EPO, as like as the short-form TPO, is due to absence of the 3'-half in the full-length TPO that confers stability both at the RNA and protein levels.

• Journal of Microbiology and Biotechnology
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• 제13권5호
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• pp.759-766
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• 2003

Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level of human thrombopoietin (hTPO) or its analog, TPO33r, were obtained by transfecting expression vectors into dihydrofolate reductase-deficient (dhfr) CHO cells and subsequent gene amplification in media containing stepwise increments in methotrexate (MTX) level such as 20, 80, and 320 nM. The parental clones with a hTPO expression level $>0.40\;{\mu}g/ml$ (27 out of 1,200 clones) and the parental clones with a TPO33r expression level $>0.20\;{\mu}g/ml$ (36 out of 400 clones) were subjected to 20 nM MTX. The clones that displayed an increased expression level at 20 nM MTX were subjected to stepwise increasing levels of MTX such as 80 and 320 nM. When subjected to 320 nM MTX, most clones did not display an increased expression level, since the detrimental effect of gene amplification on growth reduction outweighed its beneficial effect of specific TPO productivity ($q_{TPO}$) enhancement at 320 nM MTX. Accordingly, the highest producer subclones ($1-434-80^{*}$ for hTPO and $2-3-80^{*}$ for TPO33r), whose $q_{TPO}$ was 2- to 3-fold higher than that of their parental clones selected at 80 nM MTX, were isolated by limiting dilution method and were established as rCHO cel1 lines. The $q_{TPO}$ of $1-434-80^{*}\;and\;2-3-80^{*}\;was\;5.89{\pm}074\;and\;1.02{\pm}0.23\;{\mu}g/10^6$ cells/day, respectively. Southern and Northern blot analyses showed that the enhanced $q_{TPO}$ of established rCHO cell lines resulted mainly from the increased TPO gene copy number and subsequent increased TPO mRNA level. The hTPO and TPO33r produced from the established rCHO cell lines were biologically active in vivo, as demonstrated by their ability to elevate platelet counts in treated mice.

• Journal of Microbiology and Biotechnology
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• 제8권6호
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• pp.553-559
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• 1998

cDNA of human thrombopoietin (hTPO) amplified by polymerase chain reaction from a cDNA library of human fetal liver was cloned. EPO-like domains ($hTPO_{153} \;or\; hTPO_{l63})\; of\; hTPO(hTPO_{332}$) were expressed in Escherichin coli using several kinds of expression systems, such as ompA secretion, thioredoxin fusion, and the $P_L$ and T7 expression systems. To obtain $hTPO_{153}$ in soluble form, $hTPO_{153}$ cDNA was fused in-frame behind the gene encoding ompA signal sequence and thioredoxin protein. When fused with either of the genes, $hTPO_{153}$ was not expressed to the detectable level. However, a high level expression of the EPO-like domain of hTPO was obtained using the PL and T7 expression system. $hTPO_{153} \;or\; hTPO_{l63} cDNA were subcloned into the pLex and pET-28a(＋) vectors under the control of the inducible$ P_L\;T_7$ promoter, respectively. Proteins expressed using pl.ex vector and pET-28a(＋) detected in insoluble forms with an expression level of about 14% and 9% of total cellular proteins, respectively, and the level of expression was rapidly diminished in 2 h after the maximum level of expression was reached.

• 공업화학
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• 제10권8호
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• pp.1161-1168
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• 1999

3주기 전이금속(Cr~Zn)의 산화물 및 V, Mo, W의 산화물에 대하여 temperature-programmed reduction/trmperature-programmed oxidation(TPR/TPO) 실험을 통하여 그 산화-환원 특성을 조사하였다. TPO 곡선의 산화피크는 TPR 곡선의 환원피크와 비슷하거나 약간 낮은 온도에서 나타났으며, 환원피크에 비하여 온도 폭이 넓었다. 3주기 전이금속한화물의 산화 및 환원 과정의 활성화에너지는 33~149 kJ/mol 범위에 있는 반면, V, Mo, W 산화물에서는 더 컸다. 금속산화물의 산화 및 환원 과정의 활성화에너지 변화는 금속-산소 결합세기에 비례하였다. 환원(TPR) 및 산화(TPO) 과정에 대한 활성화에너지 차이(${\Delta}E_a$)가 작을수록 o-자일렌 산화반응에서 금속산화물 촉매의 활성화에너지도 작았다. 금속한화물 촉매에서 o-자일렌 산화반응은 금속산화물 표면의 산화-환원 과정을 반복하는 Mars-van Krevelen 반응 메카니즘으로 설명될 수 있음을 확인하였다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.105-108
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• 2003

To enhance the performance of a serum-free medium (SFM) for human thrombopoietin (hTPO) production in suspension cultures of recombinant Chinese hamster ovary (rCHO) cells, several low-cost hydrolysates such as yeast hydrolysate (YH), soy hydrolysate, wheat gluten hydrolysate and rice hydrolysate were tested as medium additives. Among various hydrolysates tested, the positive effect of YH on hTPO production was most significant. When 5 g/L YH was added to SFM, the maximum hTPO concentration in batch culture was 40.41 ${\mu}g/mL$, which is 11.5 times higher than that in SFM without YH supplementation. This enhanced hTPO production in YH-supplemented SFM was obtained by the combined effect of enhanced $q_{hTPO}$ and increased culture longevity. In addition YH supplementation did not affect in vivo biological activity of hTPO. Taken together, the results obtained demonstrate the potential of YH as a medium additive for hTPO production in serum-free suspension cultures of rCHO cells.

• BMB Reports
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• 제31권5호
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• pp.453-458
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• 1998

Human thrombopoietin (hTPO) was expressed to high levels in insect cells using the baculovirus expression system. Full-length hTPO cDNA containing a native signal peptide sequence was amplified by PCR from a human fetal liver cDNA library and cloned into the Autographa californica nuclear polyhedrosis virus (AcNPV) expression vector. Immunoblot analysis with antiserum against hTPO indicated that an approximately 55 kDa protein was produced in recombinant AcNPV infected insect cells. Recombinant hTPO was produced 4-fold higher in Trichoplusia ni (Tn5) cells than in Spodoptera frugiperda (Sf9) cells. with most of the hTPO produced in Tn5 cells secreted into the culture medium. Addition of tunicamycin in the culture medium resulted in the reduction of the size of hTPO to 35-38 kDa, and most of the protein remained within the cell. These results suggest that N-glycosylation of hTPO is required for the secretion of the protein into the culture medium in insect cells. hTPO produced in insect cells induced proliferation and maturation of megakaryocyte progenitors, indicating that it is in a biologically active form.

• Reproductive and Developmental Biology
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• 제32권3호
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• pp.159-166
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• 2008

본 연구는 vesicular stomatitis virus G glycoprotein (VSV-G)으로 피막이 형성되는 replication-defective MoMLV-based vector를 이용한 hTPO 헝질전환 닭의 생산에 관한 연구이다. 실험에 사용한 retrovirus vector의 구조는 hTPO 유전자의 발현 조절을 위해 internal promoter인 hCMV promoter를 이용하였으며 외래 유전자의 발현을 증가시키기 위해 woodchurk hepatitis virus posttranascriptional regulatory element (WPRE) 서열을 도입하였다. 재조합한 vector는 GP2 293 포장세포에 도입하여 virus를 생산하였으며 이 virus를 이용하여 감염시킨 여러 표적세포에서 hTPO의 발현과 생물학적 활성을 확인하였다. 재조합 hTPO의 생물학적 활성은 시판되고 있는 재조합 hTPO에 비해 우월한 것으로 확인되었다. hTPO 형질전환 닭의 생산을 위하여 1,000배 이상 고농도로 농축된 virus를 stage X 단계의 계란의 배반엽 층에 미세주입하여 대리난각 방법으로 배양하였다. 미세주입한 132개의 계란 중 21일 후에 11개의 계란에서 병아리가 부화하였으며 그중 4마리가 형질전환 개체로 확인되었다. 그러나 생산된 4마리 중 3마리가 부화 후 1개월 이내에 원인불명으로 사망하였다. 본 연구의 의의는 상업적 이용 가능성이 있는 생물학적 활성을 가진 사람의 cytokine 단백질의 대량 생산을 위한 생체 반응기로서의 형질전환 닭 개발의 시례를 제공하는데 있다.

• The Korean Journal of Physiology
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• 제5권1호
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• pp.19-22
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• 1971

The effect based upon changes of body weight and effect of X-ray irradiation on tryptophan pyrrolase (TPO) activity in liver homogenate of albino rats was studied. 1. The average TPO activity of control group of 33 rats weighing $95{\sim}172g$\;was\;2.372{\pm}0.165{\mu}$ moles kynurenine/g protein/hr. 2. Correlation between body weight of 33 rats and its TPO activity was not showed practically(r=0.011). 3. TPO activity of whole-body X-ray irradiated rats (700r) was increased about double at 3hours and increased for a period of 4 days after irradiation, but after 6 days it was decreased gradually. This result is that whole-body X-ray irradiation showed dual effect on TPO activity in rat liver.