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High-level Expression of Human Immune Interferon in Escherichia coli by using tac Promoter
곽규범,현형환,권병세,김광수,Kwack, Kyu-Bum,Hyun, Hyung-Hwan,Kwon, Byung-S.,Kim, Kwang-Soo 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.3
인간의 면역 인터페론을 코딩하는 유전자를 tac 프로모터와 rrnBT1T2 전사중지서열을 갖는 프라스미드 pKK223-3에 클론닝하여 체세포 유전자에 lacIq 제한차서열을 갖는 대장균 JM105와 체세포 유전자에 lacIq 제한자서열을 갖는 대장균 MM294와 대장균 YMC9에 각각 형질전환시켰다. 생성된 형질전환주를 immunoblotting과 생물학적 역가분석, 전기영동 그리고 전자현미경을 사용하여 확인했다. 면역 인터페론의 발현은 대장균 JM105에서 효율적으로 조절되었으며, 대장균 MM294와 대장균 YMC9에서는 IPTG의 처리없이도 생산됨을 발견하였다. 또한 면역 인터페론이 용해된 형태와 불용성 상태인 inclusion body로 생성됨을 알았다. 이때 생산성은 $9.7{\times}10^9IU/l$이며 면역 인터페론 단백질이 대장균의 전체 단백질중 약 30%에 해당함을 알았다. The cDNA for the structural sequence of mature human mmune-interferon ($IFN-{\gamma}$) was introduced into E. coli high expression vector, pKK223-3, constructed by using tac promoter and rrnBT1T2 transcription terminator region and transformed to host, E. coli JM105, which has lacIq repressor gene on chromosomal DNA, E. coli MM294, and E. coli VMC9, which have laI repressor gene. The resultant ampicillin resistant transformants were analyzed by the immunoblotting and electron microscope. $IFN-{\gamma}$, which cross-reacted with mouse anti human $IFN-{\gamma}$ serum, was produced as the soluble and the insoluble inclusion form. Their productions were efficiently regulated in E. coli JM105, but not in E. coii MM294, and E. coli YMC9 by using the IPTG.
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cDNA Cloning and Expression of a Human Interferon-$\gamma$ in E. coli
고형곤,현형환,유무영,권병세,주종광,문홍모,김광수,Koh, Hyung-Kon,Hyun, Hyung-Hwan,Yoo, Moo-Young,Kwon, Byung-S.,Jue, Chong-K.,Moon, Hong-Mo,Kim, Kwang-Soo 생화학분자생물학회 1986 한국생화학회지 Vol.19 No.3
인간의 peripheralblood lymphocyte에서 분리된 poly (A)$^+mRNA$를 사용하여 ${\lambda}gt$ 10 cDNA library를 제조하였으며, 화학적으로 합성한 oligonucleotide를 이용하여, 이 cDNA library로부터 IFN-$\gamma$ 유전자를 갖는 DNA을 분리했다. 분리된 clone 가운데 하나인 pBK04는 IFN-$\gamma$의 전체 염기서열을 갖고 있었다. IFN-$\gamma$를 E. coli내에서 발현시키기 위해 lpp promotor를 함유하고 있 는 pINIIIA3 vector를 사용하여 IFN-$\gamma$를 발현시킬 수 있는 재조합 plasmid ($pKS{\gamma}3B$)를 제조하였다. $pKS{\gamma}3B$로써 형질 변환된 bacterial cell을 antiviral activity에 의해 역가 분석을 해본 결과, 높은 정도로 IFN-$\gamma$를 발현시켰으며 이 IFN-$\gamma$는 천연의 IFN-$\gamma$에 대하여 특이성이 있는 monoclonal antibody에 의해서도 인지되었다. High copy number를 갖는 expression vector를 제조 (pHK-3) 하였으며, 이 high copy number vector를 사용하므로써 IFN-$\gamma$의 생성이 증가되는 사실을 확인하였다. A ${\lambda}gt10$ cDNA library constructed with poly $A^+$ mRNA from human peripheral blood lymphocytes was screened by a synthetic oligonucleotide having IFN-$\gamma$ sequence. One of the clones (pBK04) which hybridized to the oligomer was confirmed to have the whole IFN-$\gamma$ coding sequence. The eDNA insert was used to express IFN-$\gamma$ in E. coli under the control of lpp promoter employing pINIIIA3 vector. Cells harboring the recombinant plasmid ($pKS{\gamma}3B$) expressed high level of IFN-$\gamma$ assayed by antiviral activity, The molecule was also recognized by monoclonal antibody specific to a natural IFN-$\gamma$. An expression vector with high copy number (pHK-3) was constructed and was successfully used for the improvement of the expression of IFN-$\gamma$.
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대장균에서의 인간 Interferon - γ의 cDNA 크로닝 및 발현
고형곤,현형환,유무영,권병세,주종광,문홍모,김광수 ( Hyung Kon Koh,Hyung Hwan Hyun,Moo Young Yoo,Byung S . Kwon ) 생화학분자생물학회 1999 BMB Reports Vol.19 No.3
A λ gt10 cDNA library constructed with poly A^+ mRNA from human peripheral blood lymphocytes was screened by a synthetic oligonucleotide having IFN-γ sequence. One of the clones (pBK04) which hybridized to the oligomer was confirmed to have the whole IFN-γ coding sequence. The cDNA insert was used to express IFN-γ in E. coli under the control of lpp promoter employing pINTIIIA3 vector. Ce115 harboring the recombinant plasmid (pKS γ 3B) expressed high level of IFN-γ assayed by antiviral activity. The molecule was also recognized by monoclonal antibody specific to a natural IFN-γ. An expression vector with high copy number (pHK-3) was constructed and was successfully used for the improvement of the expression of IFN-γ
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대장균에서 인간의 면역 인터페론을 tac 프로모터를 이용하여 고도로 발현시키는 연구
곽규범,현형환,권병세,김광수 ( Kyu Bum Kwack,Hyung Hwan Hyun,Byung S . Kwon,Kwang Soo Kim ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.3
The cDNA for the structural sequence of mature human mmune-interferon (IFN-γ) was introduced into E. coli high expression vector, pKK223-3,constructed by using tac promoter and rrnBT1T2 transcription terminator region and transformed to host, E. coli JM105, which has lacIq repressor gene on chromosomal DNA, E. coli MM294, and E. coli VMC9, which have laI repressor gene. The resultant ampicillin resistant transformants were analyzed by the immunoblotting and electron microscope. IFN-γ, which cross-reacted with mouse anti human IFN-γ serum, was produced as the soluble and the insoluble inclusion form. Their productions were efficiently regulated in E. coli JM105, but not in E. coli MM294, and E. coli YMC9 by using the IPTG.
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진행암에서 Recombinant Human Interferon Alpha ( Rifn α ) 의 Phase 1 임상 및 약물동태에 관한 연구
노재경(Jae Kyung Roh),정현철(Hyun Cheol Chung),박용준(Yong Jun Park),김주항(Joo Hang Kim),김병수(Byung Soo Kim),현형환(Hyung Hwan Hyun) 대한내과학회 1989 대한내과학회지 Vol.37 No.1
N/A Fifteen patients with advanced cancers wre treated with recombinant interferon alpha (Alphaferon, Cheil Sugar Co.) by IM routes daily 5 times a week with the dose of either 3, 5, or 7´106 IU/m2 without intrapatient escalation in order to determine the tolerance and pharmacokinetics of IFN. Of the enrolled 15 patients, 12 patients were evaluable for toxicities and responses. Enrolled diseases were malignant melanoma (3), renal cell carcinoma (3), cutaneous T-cell lymphoma (3), non-small cell lung cancer (2), CML (1), breast cancer (1), stomach cancer (1), and fibrosarcoma (1). The median duration of treatment was 2 months. Of the evaluable 4 patients with the 3´105IU/m2 dose, a mild degree of flu-like syndrome in all, nausea and vomiting (severe; 1) in 3 and a mild degree of reversible elevation of hepatic transaminases was observed in 1 patient. Of the evaluable 6 patients with 5´106 IU/m2 dose, mild to moderate degree of flu-like syndrome in all, mild degree of reversible elevation of hepatic transaminases in 1, and mild degree of leukopenia in one patient was observed. Of the evaluable 3 patients with 7´108 IU/m2, severe flu-like syndrome in all, and moderate degree of leukopenia was observed in 2 patients which required dose modification. One patient with malignant melanoma showed partial response of the metastatic lymph nodes more than 12 months. Serum peak concentration of IFN was reached 4-8 hours after the IM treatment and the serum concentration was declined slowly to less than 10% of the peak concentration by 24 hours. The 24-hour urinary excretion of IFN was less than 1% of administered dose. Alphaferon was tolerable to the patients without life threatening toxicities, and the suggested MTD was 6×106 IU/m2/D with this dose schedule.
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