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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.
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.
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).
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.
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.
대장균과 포유류 세포 내에서 parkin의 발현 양상에 관한 연구
남민경,박혜민,최주연,박효진,정광철,강성만,임향숙,Nam Min-Kyung,Park Hye-Min,Choi Ju-Youn,Park Hyo-Jin,Chung Kwang Chul,Kang Seong man,Rhim Hyangshuk 한국생명과학회 2005 생명과학회지 Vol.15 No.6
E3 ligase로 알려진 Parkin은 protein quality control에서 중요한 역할을 할 뿐만 아니라, 이런 quality control system의 이상으로 나타나는 퇴행성 뇌질환에도 밀접한 연관성이었다. 이와 같이 생체의 필수적인 업무를 담당하는 Parkin의 기능을 생화학적 측면에서 연구하기 위해서는 고 순도의 단백질을 다량 정제할 수 있는 시스템이 필요하나, 아직까지 Parkin의 발현 양상과 정제법에 관한 연구가 미흡한 상태이다. 본 연구에서는 pCEX system을 이용하여 Parkin을 대장균에서 overexpression시켜 단일 스텝으로 정제할 수 있는 방법을 정립하였다. 저온의 배양조건에서 0.01 mM의 IPTC로 발현을 유도한 결과 $90\%$ 이상의 순도를 가지는 완전한 크기의 Parkin을 정제할 수 있었다. 또한, 여러 tag을 갖는 Parkin plasmid를 제작하였을 뿐만 아니라, 이들을 HEK293 세포에 transfection하여 Parkin의 발현 양상을 비교 분석하였다. 그 결과 Parkin의 N-말단에 pretense에 민감한 절단 부위가 존재한다는 사실을 확인하였다. 본 연구에서 정립한 Parkin 정제법과 포유류 세포에서 Parkin의 발현 양상에 대한 결과는 Parkin의 기질을 탐색하고,그들이 Parkin의 효소 활성 및 기능에 미치는 영향을 조사하기 위한 다양한 연구에 활용할 수 있을 것이다. Parkin, known as an E3 ubiquitin ligase, has essential role in protein quality control, and its severe dysfunction leads to neurodegenerative disorders. Human Parkin was excessively degraded when expressed in Escherichia coli under the conventional induction condition ($37^{\circ}C$ culture condition with 0.5 mM IPTG). To optimize the induction and culture conditions for recombinant human Parkin and develop a rapid method for the Parkin purification, we expressed Parkin by using PCEX system at the different culture temperatures and IPTC concentrations. The intact Parkin protein was purified to approximately $90\%$ purity with suitable amounts of protein under the optimal culture condition ($25^{\circ}C$E with 0.01 mM IPTG). Additionally, we constructed various parkin plasmids with different tagging systems and investigated their expression patterns in HEK293 cells. We found that the proteolytically sensitive site is localized within a ubiquitin-like domain of Parkin. This study developes a method for generating useful reagents to investigate biochemical properties of Parkin.
파킨슨병 환자의 뇌에서 전기영동시 서행하는 α - Synuclein 발견
최주연(Ju Youn Choi),성영모(Young Mo Sung),박효진(Hyo Jin Park),허은혜(Eun Hye Hur),김지연(Ji Youn Kim),최의열(Eui Yul Choi),박진서(Jin Seu Park),민병례(Byung Re Min),강성만(Seong Man Kang),임향숙(Hyang Shuk Rhim) 한국유전학회 2001 Genes & Genomics Vol.23 No.4
α-Synuclein is a pivotal genetic factor implicated in the pathogenesis of Parkinsons disease(PD), since pathogenicα-synuclein gene mutations occur in rare familial PD and wild-type α-synuclein appears to be a major component of Lewy bodies observed in PD. The main goal of this study was to determine whether the expression patterns of α-synuclein are differentially affected in the brains of PD, compared to normal controls. Here, we showed that new forms of the slowly-migrating α-synuclein were detected from the striatum, a region selectively affected in PD, in the brain of the patients with PD, but not from cerebellum of PD and normal controls. Moreover, these forms of α-synuclein were observed from the right brain that has a unilateral 6-hydroxydopamine lesioned nigrostriatal system in a rat model of PD. The results suggest that the new differential alteration inα-synuclein might be associated with the pathogenesis of PD.