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양문식,신영미,김영숙,Tae-Ho Kwon,Yong-Suk Jang 한국생물공학회 2003 Biotechnology and Bioprocess Engineering Vol.8 No.2
The human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene was introduced into tobacco plants. The cell suspension culture was established from leaf-derived calli of the transgenic tobacco plants in order to express and secrete a biologically active hGM-CSF. The recombinant hGM-CSF from the transgenic plant cell culture (prhGM-CSF) was identified as a yield of about 180 mg/L in the culture filtrate, as determined by ELISA. The addition of 0.5 g/L polyvinylpyrrolidone (PVP) to the plant cell culture medium both stabilized the secreted prhGM-CSF and increased the level of production approximately 1.5-fold to 270 mg/L. The biological activity of the prhGM-CSF was confirmed by measuring the proliferation of the hGM-CSF-dependent cell line, TF-1. Interestingly, the specific activity of the prhGM-CSF was estimated to be approximately 2.7 times higher than that of a commercially available preparation from E. coli.
양문식,이재화,Nguyen-Hoang Loc,Tae-Ho Kwon 한국생물공학회 2004 Biotechnology and Bioprocess Engineering Vol.9 No.1
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 KhGM-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 KhGM-CSF of 7.64 after 2 h of incubation at room temperature.
양문식,박승문,김대혁,Tae-Ho Kwon,Niti Sharma 한국생물공학회 2004 Biotechnology and Bioprocess Engineering Vol.9 No.6
A simple purification procedure of bioactive human granulocyte macrophage colony stimulating factor (hGM-CSF) secreted in rice cell suspension culture has previously been described. In this study the protein was purified to apparent homogeneity with an overall yield of 80.1% by ammonium sulfate precipitation and a single chromatographic step involving FPLC-anion exchange chromatography. The purified hGM-CSF revealed at least five glycosylated forms ranging from 21.529 kDa, and its biological activity was independent of the glycosylation pattern. This is the first purification report of recombinant hGM-CSF to apparent homogeneity from rice cell suspension cultures.
Production and Secretion of Human Interleukin-18 in Transgenic Tobacco Cell Suspension culture
양문식,김태금,Niti Sharma 한국생물공학회 2006 Biotechnology and Bioprocess Engineering Vol.11 No.2
Interleukin-18 (IL-18), otherwise known as interferon-gamma-inducing factor (IGIF), is one of several well characterized and important cytokines that contribute to host defenses. The complementary DNA (cDNA) of mature human interleukin-18 gene (hIL-18) was fused with the signal peptide of the rice amylase 1A gene (Ramy1A) and introduced into the plant expression vector under the control of a duplicated CaMV 35S promoter. The recombinant plasmid was transformed into tobacco (Nicotiana ta-bacum L. cv Havana) using the Agrobacterium-mediated transformation method. The integration of the hIL-18 gene into the genome of transgenic tobacco plants was confirmed by polymerase chain reaction (PCR) amplification and its expression was observed in the suspension cells that were derived from the transgenic plant callus by using Northern blot analysis. The hIL-18 protein was detected in the extracts of the transgenic callus and in the medium of the transgenic tobacco suspension culture by using im-munoblot analysis. Based upon enzyme-linked immunosorbant assay (ELISA) results, the expression level of the hIL-18 protein approximated 166 µg/L in the suspension culture medium. Bioassay results from the induction of interferon-g from a KG-1 cell line indicated that the hIL-18 secreted into the sus-pension culture medium was bioactive.
양문식,김태금,Maria Oszvald,Tae-Jin Kang,Barnabas Jenes,Laszlo Tamas 한국생물공학회 2007 Biotechnology and Bioprocess Engineering Vol.12 No.6
Escherichia coli heat-labile enterotoxin B subunit (LTB) can be used as a potent mucosal immunogen and immunoadjuvant for co-administered antigens. The synthetic LTB (sLTB) was modified based on plant optimized codon usage, and fused to a translation signal (the Kozak sequence) in the front of start codon and the ER retention signal, SEKDEL, in the c-terminus of sLTB gene. The sLTB and the wild-type LTB gene (wLTB) were located into plant expression vectors under the control of the wheat Bx17 HMW (High Molecular Weight) glutenin endosperm-specific promoter containing the first intron of the rice actin1 gene. Both genes were introduced into rice cells (Oryza sativa L.) via particle bombardment mediated transformation. The integration of LTB gene into the chromosome of transgenic plants was confirmed by genomic DNA PCR amplification methods. The transcription and translation of the LTB genes were demonstrated by reverse-transcription PCR (RT-PCR) and Western blot analyses, respectively. The LTB proteins produced in the seed tissues of transgenic rice showed binding affinity for GM1 ganglioside, a receptor for biologically active LTB, suggesting the plant-produced LTB are capable of forming active pentamers. The expression level of sLTB was higher than wLTB in transgenic rice plants and was up to 2.7% of the total soluble proteins of the seed tissues.