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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.
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.
방향족 아미노산에 의한 대장균 serC-aroA Operon의 발현 억제
황우길,사재훈,김경훈,임창진,Hwang, Woo-Gil,Sa, Jae-Hoon,Kim, Kyung-Hoon,Lim, Chang-Jin 한국미생물학회 1994 미생물학회지 Vol.32 No.2
대장균에서 두 가지 다른 아미노산의 생합성에 관여하는 serC 유전자와 araA유전자는 혼합 operon을 이루고 있다. SerC-aroA 혼합 operon의 발현 조절 현상을 serC-aroA-lacZ fusion plasmid pWH2를 이용하여 측정하였다. serC-aroA 혼합 operon의 발현은 L-tyrosine, L=phenulalanine 및 L-tryptophan 등 방향족 아미노산들에 의하여 억제되었다. 방향족 아미노산에 의한 억제 효과는 $tyrR^-$ 균주 혹은 $trpT^-$ 균주에서는 감소하였다. 또한, 방향족 아미노산은 cyclic AMP에 의한 이 operon의 발현 상승 효과를 감소시키기도 하였다. 이들 결과로부터 대장균 serC-aroA 혼합 operon의 발현은 방향종 아미노산들에 의해 억제된다고 추정하였다. The Escherichia coli aroA and serC genes constitute a mixed-function operon which involves in two different amino acid biosynthetic pathways. The regulation of expression of serC-aroA operon was evaluated through the use of a serC-araA-lacZ fusion plasmid pWH2. The expression of the serC-aroA operon was decreased by aromatic amino acids such as tyrosine, tryptophan, and phenylalanine. The repressible effects were diminished in E. coli tyrR of trpR strain, indicating the involvemnt of TyrR of TrpR protein in the repression. Tyrosine was competitie with cAMP in the influence on the expression of the serC-AroA operon. From these data, it was suggested that the serC-aroA operon is controlled by aromatic amino acids in a negative manner.