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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.
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).
Production of $Ser^{125}$-Interleukin-2 by Site Directed Mutagenesis
강성만,곽주원,권종범,김성완,이인영,이선복,윤혜영,함경수,한문희,나도선,Kang, Seong-Man,Kwak, Ju-Won,Kwon, Jong-Bum,Kim, Sung-Wan,Lee, In-Young,Lee, Sun-Bok,Yun, Hye-Young,Hahm, Kyung-Soo,Han, Moon-H.,Na, Doe-Sun 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.3
아미노산 서열 125번 위치의 아미노산인 시스테인을 세린으로 치환한 Inter1eukin-2를 특정 위치 변이방법에 의하여 대장균을 이용하여 생산하였다. $Ser^{125}$-IL-2는 IL-2 의존성인 배양세포에 대하여 $Cys^{125}$-IL-2와 유사한 성장촉진효과를 나타내었다. $Ser^{125}$-IL-2의 생산 수율은 $Cys^{125}$-IL-2에 비하여 1.5배 이상 높았다. Human Interleukin-2 (IL-2) posessing a serine in place of cystein at the amino acid sequence position 125 has been produced in E. coli by site directed mutagenesis. The activity of purified $Ser^{125}$-IL-2 was more than 2.5 times higher than that of $Cys^{125}$-IL-2 as determined by the growth promoting effect on IL-2 dependent cell line. The yield of production of $Ser^{125}$-IL-2 was at least 1.5 times higher as compared to that of $Cys^{125}$-IL-2.
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).
특정 위치 변이방법에 의한 Ser - interleukin - 2 - 의 생산
강성만,곽주원,권종범,김성완,이인영,이선복,윤혜영,함경수,한문희,나도선 ( Seong Man Kang,Ju Won Kwak,Jong Bum Kwon,Sung Wan Kim,In Young Lee,Sun Bok Lee,Hye Young Yun,Kyung Soo Hahm,Moon H . Han,Doe Sun Na ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.3
Human Interleukin-2 (IL-2) posessing a serine in place of cystein at the amino acid sequence position 125 has been produced in E. coli by site directed mutagenesis. The activity of purified Ser^(125)-IL-2 was more than 2.5 times higher than that of Cys^(125)-IL-2 as determined by the growth promoting effect on IL-2 dependent cell line. The yield of production of Ser^(125)-IL-2 was at least 1.5 times higher as compared to that of Cys^(125)-IL-2.
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.
국내 물류기업의 녹색물류 활성화 방안을 위한 선진 해외기업 사례연구
강성만 ( Sung Man Kang ) 한국유통경영학회(구 한국유통정보학회) 2011 유통정보학회지 Vol.14 No.1
본 연구는 국제적인 협약과 규제가 강화되고 있는 시점에 국내 물류기업의 녹색물류 활성화 방안을 모색하기 위하여, 문헌연구를 통하여 녹색물류에 대한 기존의 다양한 정의, 녹색물류의 필요성 및 환경 친화적인 물류활동을 알아보았다. 사례 분석을 통하여 각 기업들은 장비 및 기기교체, 대체연료 사용, 녹색서비스 부문에서 녹색물류 활동을 제일 많이 추진하고 있었으며, 다음으로는 정보기술을 활용한 운용 최적화, 적재 최적화, 운행노선최적화, 포장재 개선 및 재활용 등이었고, 마지막으로 녹색구매, 모달시프트, 폐기물 재활용, 에너지 소비효율 향상, 공동집배송, 에코드라이빙 순으로 나타났다. 분석 결과에 따르면, 선진 물류기업들이 단기적으로는 비용이 발생하더라도 장기적인 관점에서 장비 및 기기교체, 대체연료 사용, 서비스 부문 개선을 위한 물류정보시스템 개발 등에 막대한 투자를 하고 있으며, 이와 병행하여 업무프로세스 개선, 공급사와 고객에 대한 녹색물류 환경 조성을 통하여 전반적인 녹색물류 활동을 전개하고 있음을 알 수 있다. 따라서 국내 물류기업들의 녹색물류 활성화를 위해서는 선진 해외 물류기업의 사례에서 살펴 본 바와 같이, 녹색물류 실현을 위한 대규모 선행투자를 해야 할 것이며, 더불어 많은 투자 없이도 추진할 수 있는 업무프로세스 개선, 임직원을 포함한 공급사 및 고객에게 녹색물류 실현을 위한 지도 및 캠페인 등 녹색물류 환경 조성 등을 통하여 SCM 전반에 걸친 시너지효과를 극대화해야 할 것이다. The purpose of this paper is to recommend the way to vitalize the green logistics by logistics enterprises in Korea base on case study about the advanced foreign enterprises. The various definitions by researchers, needs and activities of environmental conscious logistics are studied through the existing literature studies. Especially, the activities of green logistics include the logistics system for environmental conscious logistics, minimization of waste materials and efficiencies of resources and energy. This case study said that the vast investments for the green logistics are required to the logistics enterprises. For example, the advanced foreign enterprises have invested capitals for the replacement of equipments and instruments, the new substitute fuels to reduce carbon dioxide, the new information system for operations and customer services. In addition, they have improved their processes including the educations and campaigns for their suppliers and customers. Therefore this paper recommends to logistics enterprises in Korea that they need to invest capitals for the replacement of equipments, instruments, the fuels. Also they are required to improve their processes and to educate their suppliers and customers to achieve the green logistics efficiently through the entire supply chain.