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Saccharomyces cerevisiae 에서 인체 Lipocortin - 1 의 유전자 발현 및 분비
손정훈,나도선,이상기 ( Jung Hoon Sohn,Doe Sun Na,Sang Ki Rhee ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.5
Human lipocortin is a phospholipase A2 inhibitory protein and known as a potential antiinflammatory agent. Thus, molecular cloning and expression of human lipocortin-1 gene was tried using a number of yeast expression vectors. cDNA of human lipocortin-1 was placed under the control of CYC1, GAL1-GAL10 and PHO5 promoters. The expression levels were varied according to the promoter strength and the plasmid copy number. It was observed that the human lipocortin-1 gene was able to be expressed under the control of all promoters employed but the highest level of gene expression was achieved by use of the divergent GAL1-GAL10 promoter. The 2 ㎛ based multicopy number plasmid showed an increased expression level by 50% compared with ARS based single copy number plasmid. To excrete human lipocortin produced in Saccharomyces cerevisiae cells into the culture medium, the pre-pro leader sequence of yeast a mating factor gene was fused with human lipocortin-1 gene. The efficient secretion of the native protein was observed with some extent of degradation in the culture medium after the post-translational processing of the fused protein.
정일엽,염영일,이상기,정태화,한문희 ( II Yup Chung,Young Il Yeom,Sang Ki Rhee,Tai Wha Chung,Moon Hi Han ) 생화학분자생물학회 1999 BMB Reports Vol.19 No.3
Interferon-γ (IFN-γ) was produced from human peripheral blood lymphocytes (Hu-PBL) by stimulating them with various kinds of mitogens. T-cell mitogens such as Con A and PHA-M were much more potent than B-cell mitogen such as PWM in producing IFN-γ from Hu-PBL. The combined treatment of TPA with these mitogens enhanced IFN-γ production ten fold compared with the single treatment of each mitogen. Both DNA synthesis and IFN-γ production yielded bell-shaped dose-response curves for a given population of Hu-PBL in response to Con A. The effect of fetal calf serum(FCS) on IFN-γ production was found to be critical: titer of IFN-γ produced was very low in the absence of FCS and reached peak point at 2% FCS, beginning to decrease precipitously with an increasing FCS concentration up to 20%. Using the relationship between IFN-γ production and mitogenic stimulation, it was found that one of factors responsible for the decrease of IFN-γ production at the high serum concentration could be the binding of Con A to serum proteins which rendered Con A to be removed from the production system.
Saccharomyces cerevisiae에서 인체 Lipocortin-1의 유전자 발현 및 분비
손정훈,나도선,이상기,Sohn, Jung-Hoon,Na, Doe-Sun,Rhee, Sang-Ki 생화학분자생물학회 1991 한국생화학회지 Vol.24 No.5
Phospholipase A2의 저해 단백질로서 항염증 치료제로 알려진 인체 lipocortin을 효모로부터 생산하기 위해 인체 lipocortin-1 유전자의 cloning 및 발현 연구를 수행하였다. 인체 lipocortin-1 cDNA를 CYC1, GALl-GALlO 및 PHO5 유전자의 promoter를 갖는 발현 vector에 cloning시킨 후 유전자의 발현을 조사한 결과 각 promoter의 강도 및 사용한 plasmid의 copy수에 따라 발현효율이 달라져 GALl-GALlO promoter와 $2{\mu}m$ 기원의 multicopy수의 plasmid를 사용했을 경우 인체 lipocortin-1 유전자의 발현효율이 가장 높음이 확인되었다. 효모 내에서 생산된 인체 lipocortin을 배지로 분비시키기 위해 효모의 ${\alpha}$ 교배인자의 pre-pro leader 배열과 인체 lipocortin-1 유전자를 융합시킨 결과 효율적인 post-translational processing을 거쳐 인체 lipocortin만이 일부 분해된 상태로서 체외로 분비됨이 관찰되었다. Human lipocortin is a phospholipase A2 inhibitory protein and known as a potential antiinflammatory agent. Thus, molecular cloning and expression of human lipocortin-1 gene was tried using a number of yeast expression vectors. cDNA of human lipocortin-1 was placed under the control of CYC1, GAL1-GAL10 and PHO5 promoters. The expression levels were varied according to the promoter strength and the plasmid copy number. It was observed that the human lipocortin-1 gene was able to be expressed under the control of all promoters employed but the highest level of gene expression was achieved by use of the divergent GAL1-GAL10 promoter. The $2{\mu}m$ based multicopy number plasmid showed an increased expression level by 50% compared with ARS based single copy number plasmid. To excrete human lipocortin produced in Saccharomyces cerevisiae cells into the culture medium, the pre-pro leader sequence of yeast a mating factor gene was fused with human lipocortin-1 gene. The efficient secretion of the native protein was observed with some extent of degradation in the culture medium after the post-translational processing of the fused protein.