Cloning and Overexpression of Escherichia Coli Hydrogenase Operon

최석정,양철학,Choi, Suk-Jung,Yang, Chul-Hak Korean Society for Biochemistry and Molecular Biol 1990 한국생화학회지 Vol.23 No.3

A set of hydrogenase-defective E. coli mutant strains have been isolated. Among them, three mutants, E. coli HD24, 43, and 50, were complemented with pHY1 previously reported (Oh et al., 1987). Another mutant strain, HD6, was transformed with E. coli chromosomal DNA library and a plasmid, pHY3, was isolated which restored hydrogenase activity in this strain. This plasmid was proved to contain two genes essential for hydrogenase activity by the complementation test of various mutants with each fragment of the insert. The two genes are contained in 2.1 kb SalI fragment of pHY3-insert. It seems that this fragment also has its own promoter. When it was expressed under the control of tac promoter, it gave two polypeptides of molecular masses, 31 and 43 kDa with a cellular content of about 10 percent. However, their increase did not correlated with the increase of hydrogenase activity. That is, they are essential components for hydrogenase activity but not sufficient. 수소발생효소의 활성이 결핍된 일련의 대장균 돌연변이체를 얻었다. 그 중에, HD24, 43 그리고 50의 세 돌연변이체는 전에 보고되었던 plasmid, pHY1에 의해 보완되었다. 다른 돌연변이체인 HD6를 대장균 염색체 DNA library로 형질변환 시킨 후, 이 돌연변이체의 수소발생효소 활성을 회복시키는 plasmid, pHY3를 얻었다. 이 plasmid는, 그 insert의 각 fragment를 이용한 보완실험에 의해, 수소 발생효소에 필수적인 두 개의 유전자를 갖고 있는 것으로 밝혀졌다. 그 두 유전자는 2.1 kb인 SalI-fragment에 포함되어 있었다. 또한 이 조각은 그 자체의 promoter를 갖고 있는 것으로 생각되며, tac promoter의 조절하에 형질발현 시켰을 때, 그 분자량이 각각 31과 43 kDa인 두 폴리펩티드를 만들었고, 그 세포내 함량은 대략 10퍼센트 정도였다. 그러나, 그 두 폴리펩티드의 양의 증가로 인해 수소발생효소 활성이 증가하지는 않는 것으로 보아, 그들은 효소활성에 필수적이기는 하나, 충분하지는 않은 것으로 생각된다.

수소발생효소의 간편한 활성 측정방법의 연구

최석정,양철학 ( Suk Jung Choi,Chul Hak Yang ) 생화학분자생물학회 1990 BMB Reports Vol.23 No.4

A spectrophotometric assay method for hydrogenase was studied. The sample solution was flushed with nitrogen or hydrogen to remove oxygen, and the reaction started. At the end of the reaction, 2`,3`,5`-tiphenyl tetrazolium chloride was reduced with reduced methyl viologen, and the concentration was determined by monitoring the absorbance at 600 nm. The absorbance increased linearly with increasing amount of enzyme in both cases of uptake and evolution assay. Alternatively, the concentration of reduced MV can be measured directly with autofill or microflow device which avoids a contact of the solution with air. This method also showed good results.

대장균 수소발생효소 유전자의 클로닝과 형질발현

최석정,양철학 ( Suk Jung Choi,Chul Hak Yang ) 생화학분자생물학회 1990 BMB Reports Vol.23 No.3

A set of hydrogenase-defective E. coli mutant strains have been isolated. Among them, three mutants, E. coli HD24, 43, and 50, were complemented with pHY1 previously reported (Oh et al., 1987). Another mutant strain, HD6, was transformed with E. coli chromosomal DNA library and a plasmid, pHY3, was isolated which restored hydrogenase activity in this strain. This plasmid was proved to contain two genes essential for hydrogenase activity by the complementation test of various mutants with each fragment of the insert. The two genes are contained in 2.1 kb Sall fragment of pHY3-insert. It seems that this fragment also has its own promoter. When it was expressed under the control of tac promoter, it gave two polypeptides of molecular masses, 31 and 43 kDa with a cellular content of about 10 percent. However, their increase did not correlated with the increase of hydrogenase activity. That is, they are essential components for hydrogenase activity but not sufficient.

교량 신축이음부에서 충돌에 따른 설계변위의 비교 및 평가

최석정(Choi Suk Jung),김동우(Kim Dong Woo),박선규(Park Sun Kyu) 대한토목학회 2000 대한토목학회논문집 A Vol.20 No.4A

TMD를 이용한 기존 보도교의 효율적 진동제어

최석정(Choi, Suk-Jung),유문식(Yoo, Moon-Sig),안상구(Ahn, Sang-Gu),박찬희(Park, Chan-Hee) 한국소음진동공학회 2003 한국소음진동공학회 학술대회논문집 Vol.2003 No.11

Molecular Cloning of Hydrogenase Gene in Escherichia coli

오경준,최석정,양철학,Oh, Kyoung-Joon,Choi, Suk-Jung,Yang, Chul-Hak Korean Society for Biochemistry and Molecular Biol 1987 한국생화학회지 Vol.20 No.2

대장균 HB101에 MNNG를 처리하고 methyl viologen 여과지법을 이용하여 hydrogenase defective 변종을 분리하였다. 대장균의 DNA를 제한효소 EcoRI으로 부분가수분해 하고 플라스미드 pBR322의 EcoRI site에 접합 시켰다. 생성된 재조합 플라스미드로 hydrogenase defective 변종 대장균 HY1을 형질전환시켰다. 그 결과 hydrogenase를 생산하는 유전자를 갖는 플라스미드 pHY1 을 얻었다. 이 플라스미드는 대장균 HY2, HY3 및 LCB850을 형질전환시키지 못하였다. 대장균의 hydrogenase 야생종, 변이종 및 pHY1-10으로 형질전환된 변이종의 crude extract를 만들어 폴리아크릴아미드 전기영동법을 이용하여 hydrogenase activity를 분석하였다. Hydrogenase defective mutants of Escherichia coli HB101 were isolated by MNNG-mutagenesis and methyl viologen filter paper screening method. E. coli DNA was digested with restriction endonuclease EcoRI and ligated into the EcoRI site of plasmid pBR322. The resulting recombinant plasmids were used to transform E. coli HY1, a mutant with a defective hydrogenase activity. Complementation of this hydrogenase mutation identified a bacterial clone carrying the gene for a E. coli hydrogenase in plasmid pHY1-10 This plasmid was not able to complement the other mutants, E. coli HY2, HY3, and LCB850. Polyacrylamide gel electrophoresis was used to analyze the hydrogenase(s) of the wild type, mutants, and mutant strains transformed with pHY1-10.

대장균의 Hydrogenase 유전자의 Cloning

오경준,최석정,양철학 ( Kyoung Joon Oh,Suk Jung Choi,Chul Hak Yang ) 생화학분자생물학회 1987 BMB Reports Vol.20 No.2

Hydrogenase defective mutants of Escherichia coli HB101 were isolated by MNNG-mutagenesis and methyl viologen filter paper screening method. E. coli DNA was digested with restriction endonuclease EcoRI and ligated into the EcoRI site of plasmid pBR322. The resulting recombinant plasmids were used to transform E. coli HY1, a mutant with a defective hydrogenase activity. Complementation of this hydrogenase mutation identified a bacterial clone carrying the gene for a E. coli hydrogenase in plasmid pHY1-10. This plasmid was not able to complement the other mutants, E. coli HY2, HY3, and LCB850. Polyacrylamide gel electrophoresis was used to analyze the hydrogenase(s) of the wild type, mutants, and mutant strains transformed with pHY1-10.

LRB의 유효설계식 산정을 위한 실험적 연구

임진석(Yim Jin Suk),최석정(Choi Suk Jung),유문식(Yoo Moon Sig) 대한토목학회 2000 대한토목학회 학술발표회 논문집 Vol.2000 No.1

Identification of a Transferrin Receptor-binding Peptide from a Phage-displayed Peptide Library

Sungil Kim(김성일),Suk-Jung Choi(최석정) 한국생명과학회 2008 생명과학회지 Vol.18 No.3

펩타이드 문고 기술을 이용하여 흑색종 세포주인 B16F10에 결합하는 펩타이드 리간드를 검색하였다. 먼저 세포 내부로 들어간 파지들을 선택하는 방법으로 두 번 검색한 후 표면에 결합한 파지들 가운데 트랜스페린 단백질을 이용하여 트랜스페린 수용체에 결합한 파지들만을 선별적으로 용출시키는 방법으로 세 번 검색하였다. 다음으로 이 두 가지 방법을 통해 선별된 파지들에 표현된 펩타이드들을 Pseudomonas exotoxin의 전이 영역과 촉매 영역에 융합시킨 재조합 독소들을 만들었다. B16F10 세포에 대한 각 재조합 독소의 활성을 측정하여 일곱 개의 클론을 선택한 후 염기서열을 분석하였다. 그 결과 그 가운데 한 클론에서 표현하는 펩타이드의 아미노산 서열이 사람의 트랜스페린과 유사한 서열을 갖는 것으로 확인되었다. 그 펩타이드를 화학적으로 합성한 후 항암제를 포함하는 리포솜에 붙여 실험한 결과 트랜스페린 수용체를 통해 치료물질을 전달할 수 있는 가능성을 지닌 것으로 평가되었다. Using a phage peptide library approach, we have isolated a peptide ligand that binds to transferrin receptor on the surface of human melanoma cell, B16F10. The library was first screened twice by recovering internalized phages and was further screened three times by competitively eluting transferrin receptor-specific phages with human transferrin among the phages bound to the cell surface. The peptides displayed by the selected phages were fused to translocation and catalytic domain of Pseudomonas exotoxin to prepare recombinant toxins. After estimating cytotoxicity of each recombinant toxin toward B16F10 cell, seven clones were selected. Sequence analysis revealed that one of the clones displayed a peptide which had a significant sequence homology with human transferrin. The peptide was chemically synthesized and was shown to be functional in delivering cytotoxic agents into B16F10 cell via interaction with transferrin receptor.

넙치와 조기의 원산지 판별을 위한 random amplified polymorphic DNA 패턴 연구

강덕진,이석근,진덕희,최석정,Kang Duk-Jin,Lee Suk-Keun,Jin Deuk-Hee,Choi Suk-Jung 한국생명과학회 2006 생명과학회지 Vol.16 No.1

본 연구에서 넙치와 조기의 원산지를 판별하기 위한 도구로 RAPD PCR 방법의 가능성을 확인하였다. 넙치는 한국의 주문진 자연산, 통영 양식산, 거제 양식산 그리고 북한 자연산을 실험에 사용하였다. 조기는 한국산과 중국산을 사용하였다. 넙치의 RAPD 패턴에서는 뚜렷하고 일관성이 있는 진단용 띠들을 쉽게 찾을 수 있었다. 조기의 경우에는 유전적인 이질성으로 인하여 각 개체의 RAPD 패턴에서는 일관성이 있는 진단용 띠를 찾기 어려웠지만 각 원산지별로 얻은 RAPD 패턴에서는 가능성이 있는 진단용 띠들을 찾을 수 있었다. The random amplified polymorphic DNA (RAPD) technique was investigated as a potential tool for the origin identification of olive flounder (Paralichthys olivaceus) and redlip croaker (Pseudosciaena polyactis). Olive flounder specimens were collected from North Korea and several locations of South Korea (Jumunjin, Tongyoung and Geoje). Fishes obtained from Tongyeong and Geoje were cultured products. Redlip croaker specimens were collected from South Korea and China. Consistent and distinct diagnostic bands were easily identified in the RAPD patterns of the olive flounder specimens. Although consistent diagnostic bands rarely appeared in the RAPD pattern of redlip croaker specimens because of their genetic heterogeneity, we were able to find potential diagnostic bands in the average RAPD pattern of each origin.