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- 저자 : 백융기 ( Chang Jin Lim (검색결과 5 건)
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Site-Directed Mutagenesis에 의한 야생형 연장된 치오레독신의 선택적 대량생성
사재훈,백혜승,이회봉,백융기,임창진,Sa, Jae-Hoon,Baek, Hye-Seung,Lee, Hee-Bong,Paik, Young-Ki,Lim, Chang-Jin 생화학분자생물학회 1992 한국생화학회지 Vol.25 No.6
An E. coli trxA mRNA contains two potential translation initiation codons, one of which initiate synthesis of a protein, called extended thioredoxin, 19 amino acid longer than thioredoxin. A mutant extended thioredoxin, which has leucine instead of methionine at its 20th position, was previously purified and charactered. In the present work, it was attempted to enhance the synthesis of wild-type extended thioredoxin without concomitant synthesis of thioredoxin. The Shine-Dalgarno (SD) sequence for the second ATG codon was changed to become non-functional by site-directed mutagenesis. The SD mutant trxA gene could normally produce wild-type extended thioredoxin but not thioredoxin. To increase the production of wild-type extended thioredoxin, the SD mutant trxA gene was put under T7 promoter. We could examine in vivo function of wild-type extended thioredoxin without interference of thioredoxin. Additionally, the selective overproduction of wild-type extended thioredoxin would make its purification much easier. 대장균 치오레독신 유전자의 mRNA는 108개의 아미노산으로 구성된 치오레독신과 그보다 19개의 아미노산이 긴 연장된 치오레독신을 합성할 수 있는 두개의 가능한 번역 개시코돈을 포함하고 있다. 두번째 개시코돈에 존재하는 methionine대신에 leucine으로 치환된 변이형 연장된 치오레독신이 정제되어 그 성질이 보고된 바 있으나, 아직 야생형 연장된 치오레독신의 특성에 관하여 알려진 바 없다. 본 연구에서는 치오레독신의 동시생성 없이 야생형 연장된 치오레독신의 합성을 증가시키기 위하여, 두번째 ATG codon에 대한 Shine-Dalgamo(SD) sequence를 site-directed mutagenesis에 의하여 기능을 못하도록 변화시켰다. 제조된 SD mutant 치오레독신 유전자를 T7 promoter vector로 subcloning하여 그 생성물을 확인하였고, 치오레독신의 간섭없이 야생형 연장된 치오레독신의 생체내 특성을 일부 파악하였다. 야생형 연장된 치오레독신의 선택적 대량생성은 이 단백질의 정제를 보다 용이하게 할 것이다.
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Site - Directed Mutagenesis 에 의한 야생형 연장된 치오레독신의 선택적 대량생성
사재훈,백혜승,이희봉,백융기,임창진 ( Jae Hoon Sa,Hye Seung Baek,Hee Bong Lee,Young Ki Park,Chang Jin Lim ) 생화학분자생물학회 1992 BMB Reports Vol.25 No.6
An E. coli trxA mRNA contains two potential translation initiation codons, one of which initiate synthesis of a protein, called extended thioredoxin, 19 amino acid longer than thioredoxin. A mutant extended thioredoxin, which has leucine instead of methionine at its 20th position, was previously purified and charactered. In the present work, it was attempted to enhance the synthesis of wild-type extended thioredoxin without concomitant synthesis of thioredoxin. The Shine-Dalgarno (SD) sequence for the second ATG codon was changed to become non-functional by site-directed mutagenesis. The SD mutant trxA gene could normally produce wild-type extended thioredoxin but not thioredoxin. To increase the production of wild-type extended thioredoxin, the SD mutant trxA gene was put under T7 promoter. We could examine in vivo function of wild-type extended thioredoxin without interference of thioredoxin. Additionally, the selective overproduction of wild-type extended thioredoxin would make its purification much easier.
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Characterization of a Mutant Extended Thioredoxin from Escherichia coli K12
임창진,최성애,백혜승,이희봉,백융기,홍순주,Lim, Chang-Jin,Choi, Sung-Ae,Back, Hye-Seung,Lee, Hee-Bong,Paik, Young-Ki,Hong, Sun-Joo 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.1
대장균 세포에 연장된 치오레독신의 존재는 site-directed mutagenesis에 의하여 확인된 바 있는데, 연장된 치오레독신은 108개의 아미노산을 갖고 있는 잘 알려진 치오레독신보다 19개 아미노산을 더 포함하고 있다. 번역시작 돌연변이 치오레독신 유전자(trxAE)는 변이형 연장된 치오레독신을 생성시키는데, 이는 두번째 ATG codon이 CTG(for leucine)로 변화되었기 때문이다. 변이형 연장된 치오레독신을 정제할 목적으로 T7 RNA polymerase/promoter system을 이용하여 $^{35}S-methionine$으로 표지되어, ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-50 gel filtration 등 정제과정에서 방사능 측정에 의하여 추적되었다. 정제된 변이형 연장된 치오레독신은 대장균 치오레독신 환원효소에 대한 친화력, 인슐린 환원과 열 안정성 등 여러 면에서 그 성질이 구명되었다. The existence of E. coli extended thioredoxin, which contains 19 amino acid residues longer than the well-known thioredoxin of 108 amino acid residues, has previously been confirmed by site-directed mutagenesis. An initiation mutant of thioredoxin gene (trxAE) gives the production of a mutant extended thioredoxin, because the second ATG codon (for methionine) was converted to CTG (for leucine). In order to purify the mutant extended thioredoxin, the protein was labeled with $^{35}S-methionine$ using T7 RNA polymerase/promoter system, and traced by radioactivity through purification steps such as ammonium sulfate fractionation, DEAE-cellulose chromatography, and Sephadex G-50 gel filtration. The purified mutant extended thioredoxin was characterized by the affinity for E. coli thioredoxin reductase, insulin reduction, and heat stability.
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임창진,김영덕,임은주,홍순주,백융기,Lim, Chang-Jin,Kim, Young-Deug,Lim, Eun-Joo,Hong, Sun-Joo,Paik, Young-Ki 생화학분자생물학회 1992 한국생화학회지 Vol.25 No.3
대장균 치오레독신 유전자의 mRNA는 두 개의 강력한 번역시작 부위를 가지고 있는데 그 중 첫번째 것은 잘 알려진 치오레독신보다 19개의 아미노산이 더 긴 단백질을 생성한다. 이전 연구에서 두번째 시작암호를 제거하여 첫번째 시작암호에 의한 번역을 확실하게 증명하였으나 그 발현 정도는 매우 낮았다. 이번 실험에서 우리는 첫번째 시작암호와 그것의 가상적인 Shine-Dalgarno(SD)의 위치 사이에 두 개의 염기를 첨가하여 SD와의 거리를 4개 염기로 만들었다. 첨가된 유전자의 조절부위를 $\beta$-유당분해효소 구조유전자와 결합시킨 뒤 $\beta$-유당분해효소의 활성측정으로서 연장된 경우의 효과를 알아 보았는데 첫번째 시작암호에 의한 번역시작이 현저하게 증가하는 것을 관찰하였다. 또한 연장된 치오레독신의 생성 증가의 효과를 여러 측면에서 조사하여 보았다. An E. coli trxA mRNA contains two potential translational initiations in the nucleotide sequence, the first one of which could initiate synthesis of a protein 19 amino acids longer than the known trxA gene product. Previously, we showed with the removal of the second ATG codon that the translation from the first ATG codon was apparently occurred, but on a very low level. In the present study, we made the insertion of two nucleotides between the first ATG codon and its presumed Shine-Dalgarno (SD) sequence, which resulted in 4 nucleotides on the distance to the SD sequence. When the effect of the extension was measured thru $\beta$-galactosidase activity after translational fusion into $\beta$-galactosidase gene, it significantly enhanced the translation for the first ATG codon. Additionally, the increased production of the extended thioredoxin was in vivo characterized in the various aspects.
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대장균 치오레독신 유전자의 두 번역 시작 부위에 관한 비교분석 : 부수적 번역 시작 부위에서의 발현 증강
임창진,김영덕,임은주,홍순주,백융기 ( Chang Jin Lim,Young Deug Kim,Eun Joo Lim,Sun Joo Hong,Young Ki Paik ) 생화학분자생물학회 1992 BMB Reports Vol.25 No.3
An E. coli trxA mRNA contains two potential translational initiations in the nucleotide sequence, the first one of which could initiate synthesis of a protein 19 amino acids longer than the known trxA gene product. Previously, we showed with the removal of the second ATG codon that the translation from the first ATG codon was apparently occurred, but on a very low level. In the present study, we made the insertion of two nucleotides between the first ATG codon and its presumed Shine-Dalgarno (SD) sequence, which resulted in 4 nucleotides on the distance to the SD sequence. When the effect of the extension was measured thru β-galactosidase activity after translational fusion into β-galactosidase gene, it significantly enhanced the translation for the first ATG codon. Additionally, the increased production of the extended thioredoxin was in vivo characterized in the various aspects.
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