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.

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).

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.

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).

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.

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.

Total Synthesis and Expression in E. coli of a Gene Coding for Human Interleukin-2

Joo, Jae Hoon,Kang, Seong Man,Song, In Sung,Kwon, Jong Bum,Han, Moon H.,Na, Doe Sun 한국산업미생물학회 1991 한국미생물·생명공학회지 Vol.19 No.3

DNA 자동합성기를 이용하여 올리고뉴클레오타이드들을 합성하였으며 이로부터 인간 인터루킨-2를 코드하는 유전자를 제조하였다. 합성 유전자의 염기서열은 대장균에서 사용빈도가 높은 코돈을 고려하여 결정하였으며, 이때 인터루킨-2의 아미노산 서열은 변화시키지 않았다. 합성된 유전자는 람다 피엘 프로모터를 사용하여 대장균에서 발현시켰다. 인터루킨-2는 대장균내에서 불용성의 침전물로 생성되었다. 생성된 인터루킨-2는 인터루킨-2 요구 배양세포주의 성장을 촉진하였다. A synthetic gene coding for human interleukin-2(IL-2) was constructed from the oligonucleotides synthesized by an automatic DNA synthesizer. The nucleotide sequence of the synthetic gene was chosen considering the preferred codons of E. coli by not changing the amino acid sequence of IL-2 polypeptide. The synthetic gene was expressed in E. coli by placing the gene under the control of the λ P_l. promoter. IL-2 was produced in the E. coli cytoplasm in the form of inclusion bodies. The recombinant IL-2 showed growth promoting activity on the IL-2 dependent cell line.