Site - directed Mutagenesis 에 의한 대장균 치오레독신 유전자의 번역시작 돌연변이체 제조 : 대장균 치오레독신 유전자 번역의 또다른 시작

임창진,임은주,김영덕,이동권,홍순주 ( Chang Jin Lim,Eun Joo Lim,Young Deug Kim,Dong Kwon Rhee,Sun Joo Hong ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.5

The mRNA produced from the E. coli thioredoxin gene (trxA) contains two potential translational initiation sites, one of which could initiate the synthesis of a protein 19 amino acids longer than the E. coli well-known thioredoxin of 108 amino acid residues. However, the expression of extended thioredoxin was not yet confirmed. To dissect translations from two initiation codons, the second ATG codon was converted to CTG by site-directed mutagenesis. The initiation-mutant thioredoxin gene lacking the second ATG codon could produce only the extended thioredoxin, which prove another translational initiation from the first ATG codon of an E. coli trxA mRNA. In vivo characteristics of the extended thioredoxin were examined in the several aspects. Just like the well-known thioredoxin, the extended thioredoxin is able to serve as a subunit of T7 DNA polymerase. In contrast, it doesn`t act as a cofactor for methionine sulfoxide reductase.

대장균 변이형 치오레독신의 성질

임창진,최성애,백혜승,이희봉,홍순주 ( Chang Jin Lim,Sung Ae Choi,Hye Seung Back,Hee Bong Lee,Young Ki Paik,Sun Joo Hong 생화학분자생물학회 1993 BMB Reports Vol.26 No.1

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 ^35S-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.

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.

흰쥐의 혈청 콜레스테롤 및 중성지방 수준에 미치는 인삼 총 사포닌의 영향

임창진,박은희,이동권,이송재,홍순근,Lim, Chang-Jin,Park, Eun-Hee,Rhee, Dong-Kwon,Lee, Song-Jae,Hong, Soon-Keun 생화학분자생물학회 1981 한국생화학회지 Vol.14 No.3

콜레스테롤을 과량 투여한 흰쥐에 정제된 인삼 총 사포닌을 용량별 및 경로 별로 투여하여 혈청 총 콜레스테롤 및 중성지방 수준에 마치는 영향을 검토하였다. 정상적인 흰쥐에서는 정제 총 사포닌의 투여 용량에 비례하여 혈청내 콜레스테롤이 감소되는 경향이 있었으며, 콜레스테롤을 과량 투여한 흰쥐에서는 총 사포닌 15 mg/kg을 경구 투여 함으로써 혈청내의 콜레스레롤양이 증가되었는데, 총사포닌 150 mg/kg을 경구투여 시에는 반대로 감소되었다. 콜레스테롤을 과량 투여한 흰쥐에 총 사포닌 15 mg 및 50 mg/kg을 복강내로 투여했을 때의 혈청내 콜레스테롤 함량은 같은 용량을 경구 투여했을 때의 콜레스테롤 함량과 비슷하였다. 콜레스테롤과 사포닌의 부분 가수분해 산물인 프로사포계닌 50 mg/kg을 경구 투여 했을 때의 혈청 콜레스테롤 수준은 콜레스테롤과 총 사포닌을 각각 500 mg/kg, 15 mg/kg을 동시에 경구 투여 및 복강내 주사 할 때와 유사하였다. 콜레스테롤과 총 사포닌을 동시에 투여한 흰쥐의 혈청내 중성지방수준은 콜레스테롤 단독 투여군에 비하여 다소 감소하였으나, 용량에 따른 차이는 인정되지 않았다. Effects of administration route and dose of ginseng total saponin on the levels of serum cholesterol and triglyceride in normal and cholesterol-administered rats were studied. The results are as follows: 1. In normal rats, serum cholesterol was decreased in proportion to the administered dose of purified total saponin. 2. In high-cholesterol administered rats, administration of total saponin 15 mg/kg (p.o.) increased serum cholesterol, but total saponin 150 mg/kg decreased it. And it was also observed that administration of prosapogenin 50 mg/kg had about the same effect as oral administration of total saponin 15 mg/kg. 3. Serum triglyceride was slightly decreased by administration of total saponin, but there was no correlationship between administered total saponin dose and serum triglyceride level.

흰쥐의 혈청 콜레스테롤 및 중성지방 수준에 미치는 인삼 총 사포닌의 영향

임창진,박은희,이동원,이송재,홍순근 ( Chang Jin Lim,Eun Hee Park,Dong Kwon Rhee,Song Jae Lee,Soon Keun Hong ) 생화학분자생물학회 1981 BMB Reports Vol.14 No.3

Effects of administration route and dose of ginseng total saponin on the levels of serum cholesterol and triglyceride in normal and cholesterol-administered rats were studied. The results are as follows 1. In normal rats, serum cholesterol was decreased in proportion to the administered dose of purified total saponin. 2. In high-cholesterol administered rats, administration of total saponin 15 ㎎/㎏ (p. o.) increased serum cholesterol, but total saponin 150 ㎎/㎏ decreased it. And it was also observed that administration of prosapogenin 50 ㎎/㎏ had about the same effect as oral administration of total saponin 15 ㎎/㎏. 3. Serum triglyceride was slightly decreased by administration of total saponin, but there was no correlationship between administered total saponin dose and serum triglyceride level.

Construction of an E. coli Initiation-mutant Thioredoxin Gene by Site-directed Mutagenesis: An Alternative Translational Initiation for E. coli trxA Gene

임창진,임은주,김영덕,이동권,홍순주,Lim, Chang-Jin,Lim, Eun-Joo,Kim, Young-Deug,Rhee, Dong-Kwon,Hong, Sun-Joo 생화학분자생물학회 1991 한국생화학회지 Vol.24 No.5

대장균 치오레독신 유전자에 대한 mRNA는 두 개의 잠재적인 번역시작 부위를 갖고 있는데, 그 중 하나는 잘 알려진 대장균 치오레독신보다 19개 아미노산이 더 많은 단백질을 합성할 수 있다. 하지만, 이 연장된 치오레독신의 존재가 아직까지는 밝혀진 바 없다. 두 시작 부위로부터의 translation을 분리 확인할 목적으로 두번째 ATG codon을 site-directed mutagenesis에 의하여 CTG로 바꾸었다. 제조된 번역시작 돌연변이체는 단지 연장된 치오레독신만을 생성하여, 대장균 치오레독신 유전자 mRNA의 또다른 번역시작을 입증하였다. 그 결과 존재가 증명된 연장된 치오레독신의 세포내 특성을 여러 측면에서 조사하였다. 이미 알려져 있는 치오레독신과 마찬가지로, 연장된 치오레독신은 T7 DNA polymerase의 한 subunit로 작용하였다. 그러나, methionine sulfoxide reductase의 cofactor로는 사용되지 않았다. The mRNA produced from the E. coli thioredoxin gene (trxA) contains two potential translational initiation sites, one of which could initiate the synthesis of a protein 19 amino acids longer than the E. coli well-known thioredoxin of 108 amino acid residues. However, the expression of extended thioredoxin was not yet confirmed. To dissect translations from two initiation codons, the second ATG codon was converted to CTG by site-directed mutagenesis. The initiation-mutant thioredoxin gene lacking the second ATG codon could produce only the extended thioredoxin, which prove another translational initiation from the first ATG codon of an E. coli trxA mRNA. In vivo characteristics of the extended thioredoxin were examined in the several aspects. Just like the well-known thioredoxin, the extended thioredoxin is able to serve as a subunit of T7 DNA polymerase. In contrast, it doesn't act as a cofactor for methionine sulfoxide reductase.

대장균 RglB 단백질의 정제 및 성질

임창진,임혜원,김경훈 ( Chang Jin Lim,Hye Won Lim,Kyung Hoon Kim ) 생화학분자생물학회 1992 BMB Reports Vol.25 No.4

The E. coli RglB protein is known to restrict methylated DNA, and 5-hydroxymethyl-cytosine-containing DNA (HMC DNA) such as non-glucosylated T-even phage DNA. The RglB protein was overexpressed in E. coli from the cloned rglB gene by T7 promoter/polymerase system. The RglB protein was purified through ammonium sulfate fractionation, DEAE-cellulose chromatography, hydroxyapatite chromatography, and Q-Sepharose chromatography. Its purity was examined on SDS-polyacrylamide gel electrophoresis. An in vitro complementation assay was used to explain the enzymatic characteristics of the purified RglB protein using E. coli HMS22 (rglA^- rglB^-) cell extract and ³H-labelled hemi-HMC M13 RF DNA.

Comparative Analysis on the Two Translational Initiation Sites of Escherichia coli Thioredoxin Gene : Enhanced Expression from the Minor Translational Initiation Site

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.

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.

항만 컨테이너 크레인의 모델링과 흔들림 억제 제어 방법

임창진(Chang-Jin Lim),최창호(Chang-Ho Choi),문상호(Sang-Ho Moon),양병훈(Byung-Hoon Yang),김흥근(Heung-Geun Kim),최종우(Jong-Woo Choi) 대한전기학회 2005 대한전기학회 학술대회 논문집 Vol.2005 No.7