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.

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.

생리활성능을 지닌 사람 인터루킨 - 1β - 재조합 단백질의 대장균의 생산

박순희,박한길,나도선,최인성,정태화 ( Soon Hee Park,Han Kil Park,Doe Sun Na,In Seong Choe,Tai Wha Chung ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.3

We produced human recombinant interleukin-1β in E. coli to study the mechanism (s) of its pleiotropic action(s), and to develop it as an anti-cancer or anti-inflammatory drug. We constructed expression vectors using lambda P_L promoter. Expression of hrIL-1β in E. coli was induced and the production of the protein was confirmed by Western blot assay. We also confirmed the presence of biological activity of recombinant IL-1β protein by demonstrating its growth promoting effect on mouse thymocytes.

Oligo - 누클레오티드 primer 를 이용한 사람 인터루킨 - 2 - cDNA의 E . coli 내 클로닝

강성만,김성완,정일엽,나도선,김지영,한문희 ( Seong Man Kang,Sung Wan Kim,Il Yub Chung,Doe Sun Na ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.1

A cDNA clone for human interleukin-2 (IL-2) was isolated by using oligonucleotides as primers for the first and the second cDNA syntheses. Total RNA was prepared from Jurkat, a human leukaemic T-cell line, cells and mRNA was isolated. To synthesize ss-cDNA, a 30 mer oligonucleotide was used as a primer in the reverse transcriptase reaction. The sequence of the oligonucleotide was complementary to the 3` end of the coding sequence of IL-2. ds-cDNA was synthesized by DNA polymerase reaction using another oligonucleotide as a primer. A partial cDNA library was prepared using the ds-cDNA and screened for the presence of IL-2 cDNA by colony hybridization using the same oligonucleotides that were used in the cDNA synthesis reactions as probes. Three out of 200 transformants showed positive signals. Analysis of these three clones by restriction enzyme mapping and nucleotide sequencing showed that all of them contained IL-2 cDNA. Our results indicated that the IL-2 cDNA was enriched in the partial cDNA library about 300 fold over the population of IL-2 cDNA in the total cDNA library reported by Taniguchi et al. (1983).

Cloning of Human Interleukin-2 cDNA in E. coli by Using Oligonucleotide Primers

강성만,김성완,정일엽,나도선,김지영,한문희,Kang, Seong-Man,Kim, Sung-Wan,Chung, Il-Yub,Na, Doe-Sun,Kim, Ji-Young,Han, Moon-Hi 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.1

사람 인터루킨-2(IL-2)의 cDNA 클론을 oligo-누클레오티드를 primer로 사용하여 분리하였다. 사람 leukaemic T-cell line인 Jurkat 세포로부터 mRNA를 분리 하였다. ss-cDNA를 합성하기 위하여 인터루킨-2를 코딩하는 mRNA의 3' 끝쪽에 상보적인 30 mer oligo-누클레오티드를 역전사 반응시 primer로 사용하였으며, ds-cDNA를 합성하기 위해서는 만들어진 ss-cDNA의 3' 끝쪽에 상보적인 oligo-누클레오티드를 primer로 사용하였다. 이 ds-cDNA를 사용하여 partial cDNA library를 만든 뒤 cDNA 합성에 사용한 oligo-누클레오티드를 probe로 사용하여 콜로니 hybridization을 하여 인터루킨-2 cDNA를 찾기위하여 screen하였다. 약 200개의 transformants 중에서 세 클론이 positive signal을 나타냈다. 제한효소지도를 작성하고 누클레오티드 염기서열을 결정함으로써 이들 세 클론이 모두 인터루킨-2 cDNA를 포함하고 있음을 밝혔다. 이 결과는 우리가 만든 partial cDNA library에 인터루킨-2 cDNA가 Taniguchi 등 (1983)이 만든 total cDNA library에 들어있는 것보다 약 300배 가량 증가되어 있음을 시사한다. A cDNA clone for human interleukin-2 (IL-2) was isolated by using oligonucleotides as primers for the first and the second cDNA syntheses. Total RNA was prepared from Jurkat, a human leukaemic T-cell line, cells and mRNA was isolated. To synthesize ss-cDNA, a 30 mer oligonucleotide was used as a primer in the reverse transcriptase reaction. The sequence of the oligonucleotide was complementary to the 3' end of the coding sequence of IL-2. ds-cDNA was synthesized by DNA polymerase reaction using another oligonucleotide as a primer. A partial cDNA library was prepared using the ds-cDNA and screened for the presence of IL-2 cDNA by colony hybridization using the same oligonucleotides that were used in the cDNA synthesis reactions as probes. Three out of 200 transformants showed positive signals. Analysis of these three clones by restriction enzyme mapping and nucleotide sequencing showed that all of them contained IL-2 cDNA. Our results indicated that the IL-2 cDNA was enriched in the partial cDNA library about 300 fold over the population of IL-2 cDNA in the total cDNA library reported by Taniguchi et al. (1983).

Recombinant Human Interleukin-2: I. Purification and Biochemical Characterization

윤혜영,최혜림,이명규,김승호,나도선,이선복,한문희,함경수,Yun, Hye-Young,Choi, Hye-Lim,Lee, Myeong-Kyu,Kim, Seung-Ho,Na, Doe-Sun,Lee, Sun-Bok,Han, Moon-H.,Hahm, Kyung-Soo 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.2

유전자 재조합 방법에 의해서 대장균으로 부터 재조합 인체 인터루킨-2 (rH IL-2, $Ser^{125}$-rH IL-2)를 과발현 시켰으며, 이 재조합 인체 인터루킨-2를 순수분리하여 그 생화학적 특성을 조사하였다. 분리 및 정제는 세포봉입체의 분리, 우레아 추출, 용해 및 젤여과 크로마토그라피 등을 사용하였으며, 정제된 인터루킨-2의 원형재현은 투석법을 사용하였다. 정제된 재조합 인터루킨-2의 순도는 전기영동 및 HPLC로 확인하였으며, 또한 결정을 얻음으로써 재확인하였다. 아미노말단 아미노산분석 및 아미노산배열순서를 일부 결정함으로서 정제된 재조합 인체 인터루킨-2가 천연 인터루킨-2와 동일한 일차구조를 갖고 있음을 확인하였다. Recombinant human interleukin-2 (rH IL-2, $Ser^{125}$-rH IL-2) was purified to apparent homogeneity from E. coli in high yield and characterized its biochemical properties for the establishment of preclinical screening system and therapeutic applications. The purification was carried out by methods involving isolation of inclusion body, urea extraction, solubilization and gel filtration chromatography. The renaturation of the product was achieved by extensive dialysis against the storage buffer. The purity was confirmed by SDS-PAGE and HPLC. Amino terminal amino acid analysis and partial amino acid sequence analysis showed that the primary structure of the recombinant protein (rH IL-2) was found to be identical with natural IL-2. The purity and the conformation of the rH IL-2 were al so confirmed by obtaining crystals of the recombinant protein.

재조합 인체 인터루킨 - 2 1 . 분리정제 및 생화학적 특성

윤혜영,최혜림,이명규,김승호,나도선,이선복,한문희,함경수 ( Hye - Young Yun,Hye - Lim Choi,Myeong Kyu Lee,Seung Ho Kim,Doe Sun Na,Sun Bok Lee,Moon H . Han,Kyung - Soo Hahm ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.2

Recombinant human interleukin-2 (rH IL-2, Ser^(125)-rH IL-2) was purified to apparent homogeneity from E. coli in high yield and characterized its biochemical properties for the establishment of preclinical screening system and therapeutic applications. The purification was carried out by methods involving isolation of inclusion body, urea extraction, solubilization and gel filtration chromatography. The renaturation of the product was achieved by extensive dialysis against the storage buffer. The purity was confirmed by SDS-PAGE and HPLC. Amino terminal amino acid analysis and partial amino acid sequence analysis showed that the primary structure of the recombinant protein (rH IL-2) was found to be identical with natural IL-2. The purity and the conformation of the rH IL-2 were also confirmed by obtaining crystals of the recombinant protein.

Overproduction of Human Interleukin-2 in E. coli

강성만,김성완,하현정,나도선,박순희,김지영,한문희,Kang, Seong-Man,Kim, Sung-Wan,Ha, Hyun-Jung,Na, Doe-Sun,Park, Soon-Hee,Kim, Ji-Young,Han, Moon-Hi 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.1

본 논문에 앞서 사람 인터루킨-2(IL-2)의 cDNA 클론을 분리하였음을 보고하였다 (강성만 등, 1987). 본 보고에서는 이 cDNA클론을 이용하여 E. coli 에서 인터루킨-2를 발현시킨 결과를 보고한다. E. coli 에서 인터루킨-2를 생산하기 위하여 플라스미드 pNK2를 만들었다. 이 플라스미드에는 인터루킨-2를 코딩하는 유전자가 람다파아지의 $P_L$ 프로모터의 조절을 받도록 클로닝 되어 있다. 온도감수성 억제유전자(${\lambda}$ cIts857)를 만드는 E. coli lysogen 균주에 플라스미드 pNK2를 transformation 시켰다. 인터루킨-2의 발현을 유도하기 위하여 배양온도를 $42^{\circ}C$로 올렸을 때 세포내에 inclusion body가 형성되었음이 관찰되었다. 재조합된 유전자를 지난 E. coli에서 합성된 단백질을 SDS-폴리아크릴아마이드 젤에서 분리하였을 때 인터루킨-2의 분자량에 해당하는 15 Kd의 단백질이 관찰되었다. 이 단백질이 인터루킨-2의 활성을 지니고 있음을 인터루킨 의존성 cell line인 MTL 세포의 성장을 촉진시키는 것으로서 확인하였다. 즉 이 15 Kd 분자량의 단백질이 E. coli에서 생산된 재조합 인터루킨-2임을 보였다. 이 인터루킨-2 단백질은 E. coli 에서 생산된 총 단백질의 약 20% 에 해당하였다. A cDNA clone for human interleukin-2(IL-2) has been isolated (Kang et al., 1987). Plasmid pNK2 was constructed in order to obtain the expression of IL-2 in E. coli. In plasmid pNK2, the coding sequence was placed under the control of phage ${\lambda}$ promoter $P_L$. An E. coli ${\lambda}$ lysogenic strain carrying a temperature sensitive repressor (${\lambda}$ cIts857) was transformed with pNK2 and the expression of IL-2 cDNA was induced by raising the temperature to $42^{\circ}C$. The inclusion bodies were observed in recombinant E. coli cells after induction. When the proteins of the recombinant cells were separated on a SDS-polyacrylamide gel, a 15Kd protein band corresponding to the M.W. of IL-2 was observed. The 15Kd protein showed IL-2 activity as determined by the growth romoting effect on a IL-2 dependent cell line MTL. Therefore, it was demonstrated that the 15Kd protein was recombinant IL-2 produced in E. coli. The recombinant IL-2 represented about 20% of the total E. coli protein.

E . coli 로 부터 사람 인터루킨 - 2 의 대량 생산에 관하여

강성만,김성완,하현정,나도선,박순희,김지영,한문희 ( Seong Man Kang,Sung Wan Kim,Hyun Jung Ha,Doe Sun Na,Soon Hee Park,Ji Young Kim,Moon Hi Han ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.1

A cDNA clone for human interleukin-2(IL-2) has been isolated (Kang et al., 1987). Plasmid pNK2 was constructed in order to obtain the expression of IL-2 in E. coli. In plasmid pNK2, the coding sequence was placed under the control of phage λ promoter P_L. An E. coli λ lysogenic strain carrying a temperature sensitive repressor (λ cIts857) was transformed with pNK2 and the expression of IL-2 cDNA was induced by raising the temperature to 42℃. The inclusion bodies were observed in recombinant E. coli cells after induction. When the proteins of the recombinant cells were separated on a SDS-polyacrylamide gel, a 15Kd protein band corresponding to the M.W. of IL-2 was observed. The 15Kd protein showed IL-2 activity as determined by the growth promoting effect on a IL-2 dependent cell line MTL. Therefore, it was demonstrated that the 15Kd protein was recombinant IL-2 produced in E. coli. The recombinant IL-2 represented about 20% of the total E. coli protein.

인간 리포코틴 - 1 유전자의 클로닝 및 대장균에서의 발현

허광래,박순희,강성만,송인성,이혜영,나도선 ( Kwahng Rae Huh,Soon Hee Park,Sung Man Kang,In Sung Song,Hay Young Lee,Doe Sun Na ) 생화학분자생물학회 1990 BMB Reports Vol.23 No.4

A cDNA clone coding for lipocortin-1 was isolated from the cDNA library of human placental mRNA. Plasmid pHT1 was constructed by inserting the coding sequence into plasmid pKK233-2, a universal expression vector containing trc promoter. Lipocortin-1 was expressed constitutively or by induction with IPTG in E. colt strain C600 or JM109 harboring pHT1. The activity of purified lipocortin-1 was demonstrated by the in vitro phospholipase A₂ inhibitory activity.