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펩타이드-Intergrase 융합 단백질의 재조합 활성
이나영,유승구 연세대학교 산업기술연구소 1996 논문집 Vol.28 No.2
Pepetide-Integrase fusion protein containing 17 additional amino acids at the amino terminal of bacteriophage Lambda Integrase was used for in vitro recombination assay and was found to have full recombination activity just as wild type Integrase. This result suggests the possibility that Integrase recombination system can be used to develop a novel peptide library system in which random peptides are displayed by DNA bound Integrase.
Plasmid DNA pBR 322의 증폭과 Hydroxyapatite Chromatography에 의한 분리
오두환,유주현,신원철,정건섭,유승구 연세대학교 산업기술연구소 1984 논문집 Vol.16 No.1
The amplification and the isolation conditions of pBR322 plasmid DNA were investigated. For the amplification of pBR322 plasmid DNA, cells from logarithmic growth phase were most effective. The optimal conditions for the amplification of pBR322 plasmid DNA in Escherichia coli GM4 were obtained as follows; 50-150 ㎍/ml of chloramphenicol concentration, 6-8 hours of incubation time in the presence of chloramphenicol. Ampicillin and tetracycline had no effect on the amplification of pBR322 plasmid DNA. Approximately 1 mg of pBR322 plasmid DNA was purified from 3.5 ℓof Escherichia coli GM4 culture broth by hydroxyapatite chromatography. The purified pBR322 plasmid DNA was expressed in Escherichia coli C600.
유승구 연세대학교 산업기술연구소 1994 논문집 Vol.26 No.2
Phosphocellulose column chromatography was performed in order to purify PRD1 DNA polymerase and terminal protein. In vitro replication assay with PRD1 natural template for each fraction showed a sole, sharp peak. The presence of PRD1 DNA polymerase and terminal protein in the peak fraction was confirmed by initiation complex formation assay. These results indicate that the PRD1 DNA polymerase and terminal protein are copurified from the crude cell extract by phosphocellulose column chromatography.
합성 기질에 의해 형성된 Lambda Site-specific Recombination 중간 대사물의 분석
이나영,유승구 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.3
Half substrate에 의해 축적된 Int-DNA 중간체를 SDS-KCI 침전과 제한효소 절단 방법으로 분석한 결과, Int-DNA 중간체는 원형이며 Int 분자가 공유결합을 하고 있슴을 알 수 있었다. 세가지의 다른 합성 기질도 Int-DNA 중간체를 축적하였다. Integrase (Int) carries out the cutting and resealing of attachment (att) site DNA via a covalent Int-DNA intermediate. A family of synthetic substrate DNAs was designed to accumulate Int-DNA intermediate. Int-DNA intermediates accumulated by half substrate was analyzed by SDS-KCI precipitation and restriction digestion. The results showed that Int-half DNA intermediate was circular and contained covalently bound Int molecule. Int-DNA intermediates were also trapped with three other kinds of synthetic substrates. Bacteriophage Lambda Intergrase(Int)는 strand exchange 위치를 절단하고 일시적으로 DNA에 결합하여 Int-DNA 중간체를 형성하며, recombination의 상대가 존재할 때 recombination을 수행한다. 본 연구에서는 특수한 기질 DNA들을 사용해서 Int-DNA 중간체(recombination 중간체)의 축적을 유도하고 이들의 성질 분석을 시도하였다.
단백질 융합 시스템을 이용한 Bacteriophage Lambda Integrase의 발현 및 정제
이나영,유승구 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.6
The lambda Integrase (Int) carries out site-specific recombination between the two partner DNA sequences, attachment P (attP) and attachment B (attB). In order to study the recombination mechanism, a large quantity of pure integrase is required. Then, we constructed an int gene inserted recombinant plasmid (pNYL3) by using the pQE31 HIS-Tag vector, and produced the fusion protein, 6xHIS-Int from the E.coli TG1 strain crrying the pNYL3 plasmid. The recombinant protein produced was purified by phosphocellulose and Ni^++-NTA affinity column chromatographies. The result of the in vitro recombination assay using the standard reaction mixture containing 6xHIS-Int and partially purified integration host factor (IHF) showed that the 6xHIS-Int tagged recombination Integrase had the full recombination activity.
Lactobacillus plantarum Bacteriophage SC 921의 Phage particle protein 및 genome의 특성
김재원,신영재,심영섭,유승구,윤성식 한국산업미생물학회 1998 한국미생물·생명공학회지 Vol.26 No.2
김치로부터 분리한 Lactobacillus plantarum bacteriophage SC 921은 M.O.I가 0.2일 경우 용균효과가 빠르게 진행됨을 알 수 있었고, SDS-PAGE를 실시하여 phage particle protein을 조사해 본 결과 4개의 major protein으로 구성되어 있는데 이들은 각각 48, 34, 32, 29 kDa으로 구성되어 있다. Exo Ⅲ로 30분간 반응시킨 후 S1 nuclease를 처리하여 DNA의 형태를 조사해 본 결과 intact DNA는 linear form의 double strand를 유전전달 물질로 가지고 있었다. 제한효소에 대한 절단 효과를 조사한 결과, Sma Ⅰ에 대해서 1개, Xba Ⅰ, Cla Ⅰ, Kpn Ⅰ, and EcoR Ⅰ에 대해서 각각 2, 4, 5, 6개의 절단부위를 가지고 있으며, Hind Ⅲ에 대해서는 절단부위가 매우 많은 것을 알 수 있었다. Hind Ⅲ를 이용하여 intact DNA의 genome size를 측정해본 결과 약 66.5 kbp정도였다. 위의 실험결과와 restriction enzyme mapping을 통해 기존에 알려진 bacteriophage B2와 비교해본 결과 숙주균주는 같으나 단백질적인 구조나 유전전달물질로 본 구조는 서로 다름을 알 수 있었다. Bacteriophage SC 921 of Lactobacillus plantarum, isolated from kimchi, showed high lytic effects at 0.2 M.O.I. level. The phage particle contained 4 major proteins (48, 34, 32, 29 kDa). Intact DNA of phage SC 921 is a double stranded linear molecule, and the genomic size is approximately 66.5 kilobase pairs (kbp). Restriction analysis of the genome showed that Sma Ⅰ gave single site cut and Xba Ⅰ gave 2 site cuts, while Cla Ⅰ, Kpn Ⅰ, and EcoR Ⅰ formed 4, 5, and 6 cuts, respectively. Hind Ⅲ digested phage DNA to many fragments. A restriction map of genomic DNA was constructed using the restiction endonuclease Kpn Ⅰ, Sma Ⅰ, and Xba Ⅰ. Bacteriophage SC 921 was compared with B2 phage which had been reported to infect Lactobacillus plantarum ATCC 8014(KCCM 11322). Bacteriophage SC 921 differs from B2 phage at least in thr size of its genome and phage particle proteins.