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- 저자 : 유명희 ( Chang Soo Kim (검색결과 6 건)
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김창수,유명희,Kim, Chang-Soo,Yu, Myeong-Hee 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.1
Three recombinant vectors were constructed to express the X open reading frame of hepatitis B virus in E. coli. All the expression vectors carried in common the tac promoter, sequence encoding X protein from 10th amino acid residues, and the lacZ sequence fused in frame with X. The variation was located at the 5' coding region of the fusion gene. Upon induction, the X-${\beta}$-galactosidase fusion proteins encoded by pTXZ2 plasmid only accumulated substantially inside the cells. Analysis of predicted mRNA secondary structures of the X-lacZ fusion genes in three different plasmids suggest that accesibility to ribosome is important to maximal expression. Furthermore we found that the steady state level of X-lacZ mRNA in each recombinant cells was correlated with the level of the fusion protein expressed. These observations are consistent with a model that appropriate binding of ribosome is important for protecting mRNA from degradation. B형 간염바이러스의 X ORF를 대장균에서 발현시키기 위해 세 개의 발현 벡터를 제조하였다. 모든 벡터들은 공통적으로 택 프로모터 서열, X 단백질을 열번 째 잔기부터 코우드하는 염기서열, 그리고 X 유전자와 프레임이 맞도록 연결된 lacZ 염기서열을 지니고 있으며, 서로 다른 부위는 X-lacZ 융합유전자의 5' 코딩 서열이다. 이들 플라스미드는 벡터로부터 융합단백질의 생산을 유도한 결과, pTXZ2를 함유하는 세포만이 다량의 X-베타갈락토시다제 융합단백질을 생산하였다. 이들 벡터의 X-lacZ 유전자에 의해 코드되는 mRNA의 2차 구조를 분석한 결과, mRNA 의 5' 코딩서열 부위에 리보좀과 결합할 수 있는 열려진 구조가 존재하는 것이 발현 최대화에 중요함을 암시하고 있다. 뿐만 아니라 이들 재조합 균주내의 X-lacZ mRNA 양은 발현된 단백질 양과 상관관계가 있는 것을 알 수 있었는데, 이러한 결과는 mRNA의 리보좀 결합력이 mRNA가 세포 안에서 분해되는 것으로부터 보호한다는 모델과 일치한다.
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ard 타입 B 형 간염 바이러스의 유전자 X 부위의 핵산서열 결정 및 발현
김창수,유명희 ( Chang Soo Kim,Myeong Hee Yu ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.4
Nucleotide sequence encompassing the X open reading frame (ORF) of a hepatitis B virus subtype adr was determined and was compared with other reported sequences Sequences between various isolates were highly conserved with about 94-98 % homology. Variations in nucleotide sequence appeared throughout the whole ORF. On the contrary, most of the amino acid substitions occurred in residues 30-40, the region at which no secondary structure was predicted. The X protein was successfully expressed in E. coli by a fusion to the C-terminus of a truncated β-galactosidase. The yield of the fusion protein was 15 % of total cellular proteins.
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대장균에서 B 형 간염바이러스 X 단백질의 발현에 필요한 5 ' - 코딩 서열
김창수,유명희 ( Chang Soo Kim,Myeong Hee Yu ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.1
Three recombinant vectors were constructed to express the X open reading frame of hepatitis B virus in E. coli. All the expression vectors carried in common the tac promoter, sequence encoding X protein from 10th amino acid residues, and the IacZ sequence fused in frame with X. The variation was located at the 5` coding region of the fusion gene. Upon induction, the X-β-galactosidase fusion proteins encoded by pTXZ2 plasmid only accumulated substantially inside the cells. Analysis of predicted mRNA secondary structures of the X-IacZ fusion genes in three different plasmids suggest that accesibility to ribosome is important to maximal expression. Furthermore we found that the steady state level of X-lacZ mRNA in each recombinant cells was correlated with the level of the fusion protein expressed. These observations are consistent with a model that appropriate binding of ribosome is important for protecting mRNA from degradation.
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Inclusion Body Formation of Recombinant ${\beta}$-Galactosidase Fusion Protein in Escherichia coli
김창수,유명희,Kim, Chang-Soo,Yu, Myeong-Hee 생화학분자생물학회 1990 한국생화학회지 Vol.23 No.2
대장균 JM109 균주에서 B형 간염 바이러스의 X 부위가 연결된 재조합 융합베타갈락토시다제를 발현시켰을 때 베타갈락토시다제 효소생산은 온도에 민감하게 반응하였다. 높은 온도에서는 효소생산량이 저하되었는데, 이는 융합폴리펩티드의 생산이 감소되어서가 아니라 폴리펩티드들이 세포내에서 응집되었기 때문이다. 하지만 낮은 온도 $(17^{\circ}C)$에서 제대로 폴딩된 단백질은 높은 온도$(37^{\circ}C)$에서도 활성을 그대로 유지하였다. 세포내에서 일단 생성된 단백질 응집물은 배양온도를 낮추어도 활성이 있는 상태로 돌아갈 수 없었다. 이러한 결과로 미루어 볼 때 응집현상은 native 상태의 단백질이 열에 의한 변성에 의해 유도되는 것이라기 보다는 높은 온도에서 폴리펩티드폴딩이 제대로 진행되지 못한 결과라 할 수 있겠다. Recombinant Escherichia coli JM109 cells expressing ${\beta}$-galactosidase fused with X open reading frame of hepatitis B virus exhibited temperature sensitive production of ${\beta}$-galactosidase. Decrease in activity at high culture temperature $(37^{\circ}C)$ was not due to the decrease in fusion polypeptide production per se, but rather due to aggregation of the fusion polypeptides within the cells. However once the fusion polypeptides folded into proper conformation at low culture temperature $(17^{\circ}C)$, they maintained the activity even at high temperature $(42^{\circ}C)$. Aggregates of the fusion polypeptides did not convert to active form when the culture temperature was lowered. The results indicate that aggregation is not a consequence of thermal denaturation of native structure but of incorrect folding and assembly of the polypeptides at high culture temperature.
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김창수,유명희,Kim, Chang-Soo,Yu, Myeong-Hee 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.4
adr 타입 B형 간염 바이러스의 유전자 X의 핵산서열을 결정하고 보고된 다른 X의 핵산서열과 비교하였다. 결정된 핵산서열은 다른 타입의 서열과 비교하여 94-98% 정도의 유사성을 갖고 있었으며, 핵산서열의 염기치환은 전구간에 걸쳐 퍼져있었다. 핵산서열로부터 유추된 아미노산서열들을 비교하였을 때 아미노산 잔기의 치환은 잔기번호 30-40 사이에 특히 많이 분포되어 있다. 단백질 2차구조 예측에 의하면 부위의 아미노산 잔기는 특정한 2차구조를 지니고 있지 않음이 밝혀졌다. 이 재조합된 X 단백질은 절단된 $\beta$-galactosidase의 카르복실발단에 융합된 상태로 대장균에서 성공적으로 발현되었다. 그 발현수율은 전체 단백질의 15% 도를 차지하고 있다. Nucleotide sequence encompassing the X open reading frame (ORF) of a hepatitis B virus subtype adr was determined and was compared with other reported sequences. Sequences between various isolates were highly conserved with about 94-98% homology. Variations in nucleotide sequence appeared throughout the whole ORF. On the contrary, most of the amino acid substitions occurred in residues 30-40, the region at which no secondary structure was predicted. The X protein was successfully expressed in E. coli by a fusion to the C-terminus of a truncated, $\beta$-galactosidase. The yield of the fusion protein was 15% of total cellular proteins.
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Inclusion Body Formation of Recombinant β - Galactosidase Fusion Protein in Escherichia coli
김창수,유명희 생화학분자생물학회 1994 BMB Reports Vol.23 No.2
Recombinant Escherichia coli JM109 cells expressing β-galactosidase fused with X open reading frame of hepatitis B virus exhibited temperature sensitive production of β-galactosidase. Decrease in activity at high culture temperature (37℃) was not due to the decrease in fusion polypeptide production per se, but rather due to aggregation of the fusion polypeptides within the cells. However once the fusion polypeptides folded into proper conformation at low culture temperature (17℃), they maintained the activity even at high temperature (42℃). Aggregates of the fusion polypeptides did not convert to active form when the culture temperature was lowered. The results indicate that aggregation is not a consequence of thermal denaturation of native structure but of incorrect folding and assembly of the polypeptides at high culture temperature.
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