• BMB Reports
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• v.31 no.5
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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.

• Proceedings of the PSK Conference
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• 2003.10b
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• pp.237.2-237.2
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• 2003

DWP40458 is a novel human thrombopoietin mutein with two additional N-linked glycosylation site. The thermal stability of DWP40458 in both solution and lyophilized form was studied in the temperature range of 4-50$^{\circ}C$, compared with recombinant human TPO (rhTPO). When the aggregation or degradation pattern of DWP40458 and rhTPO solution was characterized by using SDS-PAGE, gel permeation chromatography (GPC) and reverse phase HPLC, it was found that thermostability of DWP40458 was significantly different to rhTPO in the temperature at 25, 40, 40, 50$^{\circ}C$. (omitted)

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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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.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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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.

• Journal of Life Science
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• v.17 no.11
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• pp.1497-1504
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• 2007

To investigate the function and secretion of human thrombopoietin (TPO) in mammalian cells, hTPO cDNA was cloned using human liver cDNA, and recombinant hTPO (rec-hTPO) was produced in CHO cell lines. In addition, six N-linked glycosylation sites were substituted for Ala to elucidate the role of each carbohydrate chain. To analyze the biological activity, rec-hTPO protein was injected subcutaneously. Blood was withdrawn for platelet determination. The metabolic clearance rate (MCR) was also analyzed at the 1, 4, 10 and 24 hr after tail vein injection. Wild-type TPO (WT) was efficiently secreted into the medium. However, a hTPO mutant with 116 deleted nucleotides detected by PCR cloning was not secreted. The N-linked glycosylation sites had nearly the same expression quantity as rec-hTPO WT apart from mutants 3 and 4. The glycosylation site of mutant 4 appeared to be an indispensable site for hTPO secretion. Also characterized was the biological activity through an injection with rec-hTPO (10 ng) to ICR mice (7 weeks). The result of the blood analysis showed a considerable increase in the platelet number six days after He injection. To analyze the pharmacokinetics, rec-hTPO was injected into the tail vein (5 ng). The result was 200 pg/ml 1hr after this injection. Following this, it dramatically decreased and virtually disappeared 10 hours after the injection. Thus, rec-hTPO may be a treatment for thrombopenia by the production of the high active rec-hTPO. In addition, hTPO can permit the development of potent new analogues that stimulate the platelet value.

• Proceedings of the Korean Biophysical Society Conference
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• 1997.07a
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• pp.43-43
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• 1997

Platelet production in blood is regulated by a lineage specific humoral factor called thrombopoietin (TPO). The amino terminal domain of TPO (TPO-N) has a sequence homology to erythropoietin (EPO) and is responsible for the signal transduction mediated by the TPO receptor, c-mpl.(omitted)

• Journal of Microbiology and Biotechnology
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• v.9 no.3
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• pp.318-322
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• 1999

A new baculovirus transfer vector (pPGP404) was constructed to increase the expression level of human thrombopoietin (hTPO) in insect cells. In pPGP404, hTPO was cloned next to the AcNPV polyhedrin-gp64 dual promoter and the leader sequence of hTPO was substituted with that of gp64. A recombinant baculovirus, AcPGP404, was constructed by using pPGP404 as a transfer vector. hTPO was expressed in AcPGP404-infected TN5 cells and it was observed that the expression levels of hTPO in TN5 cells increased three-fold ($6.0 {\mu}g/ml^{-1}$) compared to the level expressed under the control of the polyhedrin single promoter. These results indicate that the polyhedrin-gp64 dual promoter system would be useful for expression in large quantities of recombinant proteins in insect cells.

• KSBB Journal
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• v.19 no.5
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• pp.341-347
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• 2004

Several gene delivery therapies are being developed for treatment of serum protein deficiency. EPO is one of the most promising therapeutic agent for this treatment which is currently being investigated in depth. This study has the ultimate purpose of improving the gene delivery system for an increase of red blood cell production. A plasmid DNA was constructed smaller than other plasmids for an increase in penetration into animal cells, and two genes were cloned into each vector as a co-delivery system to express erythropoietin, and interluekin-3 or thrombopoietin, which can act on erythroid cell, thus activating hematopoiesis synergically. This co-delivery system has an advantage of decreasing the labour required for industrial production of DNA vaccine. A new plasmid vector, pVAC, in size 2.9 kb, was constructed with the essential parts from PUC 19 and pSectagB, which is much smaller than other plasmid vector and is the size of 2.9 kb. Co-delivery system was constituted by cloning human erythropoietin with each of human interluekin-3 gene or human thrombopoietin gene into both pVAC and pSectagB. As a result, the transfection efficiency of pVAC was higer than that of pSectagB in vitro, and hematocrit level of the mice injected with pVAC is higher than that of other mice. And co-delivery system, made of several plasmid DNAs, was expressed in vitro.

• Reproductive and Developmental Biology
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• v.32 no.3
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• pp.159-166
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• 2008

We report here the generation of transgenic chickens that produce human Thrombopoietin (hTPO) using replication-defective Moloney murine leukemia virus (MoMLV)-based vectors packaged with vesicular stomatitis virus G glycoprotein (VSV-G). For the retrovirus vectors, we used hCMV (human Cytomegalovirus) internal promoter to drive the hTPO gene. After confirming the expression of the hTPO gene in various target cells, the concentrated solution of recombinant retrovirus was injected beneath the blastoderm of non-incubated chicken embryos (stage X). The biological activity of the recombinant hTPO in target cell was significantly higher than its commercially available counterpart. Out of 132 injected eggs, 11 chicks hatched after 21 days of incubation and 4 hatched chicks were found to express vector-encoded hTPO gene. However, 3 out of the 4 transgenics died within one month of hatching. The major significance of this study is that it is one of the very few successful reports on the production of transgenic chickens as bioreactors aiming mass production of commercially valuable and biological active human cytokine proteins.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.6
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• pp.806-813
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• 2010

Animal bioreactors have been regarded as alternative tools for the production of limited human therapeutic proteins. The mammary glands of cattle are optimal tissues to produce therapeutic proteins that cannot be produced in large amounts in traditional systems based on microorganisms and eukaryotic cells. In this study, two knock-in vectors, pBCTPOKI-6 and pBCTPOKI-10, which target the hTPO gene on the bovine beta-casein locus, were designed to develop cloned transgenic cattle. The pBCTPOKI-6 and pBCTPOKI-10 vectors expressed hTPO protein in culture medium at a concentration of 774 pg/ml and 1,867 pg/ml, respectively. Successfully, two targeted cell clones were obtained from the bovine fibroblasts transfected with the pBCTPOKI-6 vector. Cloned embryos reconstructed with the targeted nuclei showed a lower in vitro developmental competence than those with the wild-type nuclei. After transfer of the cloned embryos into recipients, 7 pregnancies were detected at 40 to 60 days of gestation, but failed to develop to term. The results are the first trial for targeting of a human gene on the bovine milk protein gene locus, providing the potential for a large-scale production of therapeutic proteins in the animal bioreactor system.