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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.
Production of Interferon-$\gamma$ from Human Peripheral Blood Lymphocytes
정일엽,염영일,이상기,정태화,한문희,Chung, Il-Yup,Yeom, Young-Il,Rhee, Sang-Ki,Chung, Tai-Wha,Han, Moon-Hi 생화학분자생물학회 1986 한국생화학회지 Vol.19 No.3
인체혈액에서 분리한 임파구세포를 PHA-M, Con A, PWM등의 mitogen으로 자극시켜 여러가지 조건하에서 감마-인터페론을 생산하였다. 유도물질로 사용된 T-세포 mitogen인 PHA-M이나 Con A가 B-세포 mitogen 인 PWM보다 감마-인터페론 유도에 훨씬 효율적이었으며 이로부터 임파구세포 중에서 T-세포가 감마-인터페론을 생산하는 주요세포원 임을 확인하였다. 감마-인터페론 생산시 Con A의 적정농도는 $5-10\;{\mu}g/ml$이었다. 유도개시 약 48시간 경과 후 인터페론이 최대로 생산되었고 그 이후에도 인터페론의 활성은 감소되지 않았다. Phorbolester인 TPA를 첨가할 경우 Con A 또는 PHA-M 단독으로 인터페론을 유도시킨 것보다 10배 이상의 감마-인터페론의 생산이 증가되었다. 생산배양액에 첨가되는 혈청의 농도가 인터페론의 생산에 마치는 영향은 매우 극적이어서 태아 송아지 혈청농도 2%에서 최대로 생산되었고 농도가 증가함에 따라 생산역가는 급격히 떨어졌다. 혈청 대신 사용한 알부민 단백질도 비슷한 효과를 나타냈다. 혈액중에 임파구세포에 처리된 Con A가 DNA 합성을 촉진시키는 성질을 이용하여 $^3H$-thymidine 표식으로 cpm을 측정해 본 결과, 이와같은 현상은 10-20%의 높은 혈청농도에서 Con A가 과량의 혈청단백질과 흡착하여 실제적인 Con A 농도를 적정농도 이하로 떨어뜨리기 때문인 것으로 나타났다. Interferon-$\gamma$(IFN-$\gamma$) 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-$\gamma$ from Hu-PBL. The combined treatment of TP A with these mitogens enhanced IFN-$\gamma$ production ten fold compared with the single treatment of each mitogen. Both DNA synthesis and IFN-$\gamma$ 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-$\gamma$ production was found to be critical: titer of IFN-$\gamma$ 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-$\gamma$ production and mitogenic stimulation, it was found that one of factors responsible for the decrease of IFN-$\gamma$ 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 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.