• Biotechnology and Bioprocess Engineering:BBE
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• 제1권1호
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• pp.9-12
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• 1996

In order to produce the single chain precursor of a novel human insulin analogue, (B30-Homoserine) insulin, the fermentative behaviors of Escherichia coli JM103 were studied, which harbors pKBA plasmid carrying a hybrid gene in which the gene for a single chain precursor was fused with lacZ gene under tac promoter. The maximal induction of gene expression was achieved when more than 0.05 mM of isopropyl-$\beta$-D-thiogalactopyranoside(IPTG) was supplemented to fermentation medium after 4 h cultivation of E. coli, and followed by longer than 2-h fermentation. The hybrid protein of the single chain insulin precursor was isolated from cytoplasmic inclusion bodies by dissolving in 8M urea solution, and purified through DEAE-Sephacel and Sephadex G-200 column chromatographies with a recovery of 35%. The finally purified hybrid protein showed a single band on sodium dodecyl sulfate-polyacrylamide gel.

• Journal of Microbiology
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• 제38권3호
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• pp.145-149
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• 2000

GroEL is a typical molecular chaperone. GroEL synthesis patterns at various culture temperatures in Escherichia coli were investigated in this study. No significant differences in the amount of GroEL produced from the chromosome were found at 30 and 37$^{\circ}C$. However, GroEL production increased 3.4-fold at 42$^{\circ}C$. GroEL synthesis was not transient but continuous at 42$^{\circ}C$, although most heat shock gene expression is known to be transient. To understand the role of the groEL structural gene, a groE promoter-lacZ fusion was constructed. Interestingly , while transcriptional fusion is not thermally inducible, it is inducible by ethanol, suggesting that the secondary structure of the groEL transcript is involved in thermal regulation of the groEL gene. Secondary structures of groE mRNA at 37 and 42$^{\circ}C$ were compared using the computer program RNAdraw. Distinct structures at the two temperatures were found, and these structures may be related to a high level of GroEL expression at 42$^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• 제13권6호
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• pp.976-983
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• 2003

Seven Escherichia coli cells defective with aerobic growth were isolated by the insertion of ${\lambda}placMu53$, a hybrid bacteriophage of ${\lambda}$ and Mu, which created a transcriptional fusion to lacZY. These insertion mutant cells were tested on an XG ($5-bromo-4-chloro-3-indolyl-{\beta}-D-galactopyranoside$) medium for anaerobic expression of lacZ by fusion to a promoter. The chromosomal DNA from these strains were digested by EcoRI, and the EcoRI fragments that contained the fused gene and lacZ sequence were identified by Southern hybridization, using lacZ containing plasmid as a probe. The EcoRI fragment from each strain was cloned and sequenced. The sequence data were compared with the GenBank database. The mutated gene of three strains, CYT4, CYT5, and OS11, was found to be identical, and it was nrdAB that encoded ribonucleoside diphosphate reductase. The gene nrdAB was at min 50.5 on the Escherichia coli linkage map and 2,348,084 on the physical map, and is involved in hemAe-related reduction-oxidation reaction. OS6 and OS14 mutant strains had insertion at min 8.3 and the mutated gene was hemB. The hemB encodes 5-aminolevulinate dehydratase or porphobilinogen synthase. The OS3 mutant had insertion in cydB at min 16.6. The cydAB encodes cytochrome d oxidase. In the case of OS1, the fusion was made with sucA, the E1 component of ${\alpha}-ketoglutarate$ dehydrogenase.

• Journal of Microbiology and Biotechnology
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• 제17권12호
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• pp.2046-2055
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• 2007

Wild-type V. vulnificus cannot grow using lactose as the sole carbon source or take up the sugar. However, prolonged culture of this species in media containing lactose as the sole carbon source leads to the generation of a spontaneous lactose-utilizing (LU) mutant. This mutant showed strong ${\beta}$-galactosidase activity, whereas the wild-type strain showed a barely detectable level of the activity. A mutant with a lesion in a gene homologous to the lacZ of E. coli in the bacterium no longer showed ${\beta}$-galactosidase activity or generated spontaneous LU mutants, suggesting that the lacZ homolog is responsible for the catabolism of lactose, but the expression of the gene and genes for transport of lactose is tightly regulated. Genetic analysis of spontaneous LU mutants showed that all the mutations occur in a lacI homolog, which is located downstream to the lacZ and putative ABC-type lac permease genes. Consistent with this, a genomic library clone containing the lad gene, when present in trans, made the spontaneous LU mutants no longer able to utilize lactose as the sole carbon source. Taken together with the observation that excessive amounts of exogenously supplemented possible catabolic products of lactose have negative effects on the growth and survivability of V. vulnificus, we suggest that V. vulnificus has evolved to carry a repressor that tightly regulates the expression of lacZ to keep the intracellular toxic catabolic intermediates at a sublethal level.

• BMB Reports
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• 제28권5호
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• pp.458-463
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• 1995

Two apparent rho-independent transcription terminator structures within the coding sequence of aceK have been destroyed to access their roles in the differential expression between aceA and aceK in the glyoxylate bypass operon of E. coli. The effect of mutations on the expression of aceK was evaluated in two different ways: one by maxicell labeling and the other by lacZ fusion gene construction. The maxicell labeling experiment with the mutant operon clones has failed, like that of the wild type operon clone, to visibly show isocitrate dehrogenase (IDH) kinase/phosphatase, the product of aceK, on the autoradiogram of a protein gel. When the same mutations were introduced into an aceK::lacZ fusion gene to quantitatively evaluate the mutational effect, the activity of ${\beta}-galactosidase$ in neither of the mutant versions of the fusion gene was elevated significantly enough to explain the degree of polarity observed in this region. Thus, we conclude that neither of these intragenic, apparent rho-independent transcription terminator structures, which have long been suspected as a major determinant in the down regulation of aceK, really act as a premature transcriptional terminator.

• 한국미생물·생명공학회지
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• 제17권4호
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• pp.334-342
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• 1989

Bacillus stearothermophilus의 cytidine deaminase (cytidine/2'-deoxycytidine aminohydrolase：EC 3.5.4.5)를 코딩하는 cdd 유전자를 E. coli cdd$^-$ 결손변이주를 cloning host로 하여 3-10Kbp의 B. stearothermophilus DNA 단편으로부터 shot gun 법으로 클로닝하였다. 고 복제수 플라스미드 pBR322 의 PstI 부위에 3.0Kb의 B. stearothermophilus DNA 단편을 함유한 pJSC101이 cdd$^+$와 tetracy-line 내성으로서 cloning되었으며, 이어서, 결실 및 subcloning을 연속 수행한 결과 약 1.35kbp의 Eco RI$_1$/PstI$_2$단편이 동일 부위의 pBR322에 삽입된 cdd 양성의 pJSC201을 얻었다. Mini 세포 실험결과, 이 단편에서 합성되는 polypeptide는 약 33 KDa이었기에 이 polypeptide가 cytidine deaminase 로 추정되었다. 또한 이 단편에 함유한 550bp의 EcoRI/AvaI 부분을 lacZ 프로모터 영역에 삽입한 경우 프로모터 활성을 나타내었기에 이 단편의 Eco RI 부위에서 PstI부위로 cdd 유전자가 전사됨을 알 수 있었다. B. subilis와 E. coli에서 발현이 가능한 shuttle vector에 cdd가 함유된 단편을 삽입한 후 이를 양세포에서 동시 발현시켰을 때 B. subtilis에서 발현시킨 경우가 E. coli에서 보다높은 cytidine deaminase 활성을 나타내었으며 이 유전자는 B. subtilis 에서도 E. coli에서와 같이 안정하게 유지됨을 알 수 있었다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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• pp.619-622
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• 2000

For the production of $B^{30}-homoserine$ human insulin precursor, four types of fusion peptides LacZ, MBP, GST, and His-tagged sequence were studied in this work. Recombinant E. coli JM 103 and E. coli JM 109 containing fusion peptides were cultivated at $37^{\circ}C$ for 1hr, and gene expression was occurred when 0.5mM of isopropyl-D-thiogalactoside(IPTG) was added to the culture broth, and followed by longer than 4hr fermentation respectively. DEAE-Sphacel and gel filtration chromatography, amylose and glutathione-Sepharose 4B affinity chromatography, and nickel-affinity chromatography system were employed as purification of $B^{30}-homoserine$ human insulin precursor. Recovery yields of His-tagged, LacZ, GST, and MBP fused $B^{30}-homoserine$ human insulin precursor resulted in 47%, 20%, 20%, and 18%, respectively.

• 미생물과산업
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• 제17권1호
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• pp.2-9
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• 1991

All of four genes are cloned and DNA sequence analysis have now revealed that these four genes encode a family of proteins with similar amino acid sequence. These proteins show no extensive similarities to any known proteins (Haarer et al., 1991). Among them, CDC3 gene is fused with E. coli lacZ and trpE genes and antibodies against the CDC3 gene product are produced. These antibodies are used to check the localization of this product to the vicinity of the 10 -nm filaments in the mother-bud neck.

• Applied Biological Chemistry
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• 제39권6호
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• pp.443-448
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• 1996

xylA 유전자의 프로모터상에서 조절양상을 조사하기 위하여 xylA 유전자의 프로모터(Pxyl)와 lacZ 유전자를 연결한 Pxyl-lacZ 융합 유전자를 제작하여 xylose에 의한 ${\beta}-galactosidase$ 생산의 조절양식을 조사하였다. xylA 프로모터 부위를 분리하여 lac 프로모터가 없는 고복제수의 lac 오페론 백터인 pMC1403에 클로닝시켜 pMCX191을 제작하여 reading frame에 변화가 없는 Pxyl-lacZ 융합 유전자를 만들었으며 이 벡터에서 Pxyl-lacZ 단편을 분리한 후 저복제수 벡터인 pLG339에 클로닝시켜 pLGX191을 제작하였다. 상기 플라스미드들을 xylA 변이주인 DH77에 형질전환시켜 Pxyl-lacZ 융합 유전자에서 ${\beta}-galactosidase$의 발현조절을 조사한 결과 xylose 농도에 따른 유도, glucose에 의한 발현억제 및 cAMP에 의한 억제해제 양상 등이 염색체상의xylA 유전자의 발현조절과 같은 경향을 나타내었다. pMCX191과 pLGX191을 이용하여 유전자 투여 량 효과를 본 결과도 복제수에 따른 차이가 크지 않았다. xylA 프로모터 부위내 조절영역를 추정하기 위해 구조유전자 상류 -209 bp를 포함한 xylA 유전자를 pUC19에 클로닝시킨 pUX30에서 프로모터 부위가 부분결손된 벡터들을 제작하여 결손부위의 염기서열을 확인하였다. 이들 부분결손 xylA 프로모터를 가진 xylA 유전자에서 xylose isomerase의 발현을 조사한 결과, 번역 개시점에서 -166 bp 이상의 영역을 결손시킨 pUX31과 pUX32의 경우pUX30과 비슷한 발현 양상을 보인 반면 -120 bp 이상의 영역을 결손시킨 pUX33과 pUX34에서는 모벡터에 비해 약 30% 수준의 발현을 보였다. 또한 pUX33과 pUX34에서는 xylose 유도시 cAMP에 의한 발현 촉진효과도 볼 수 없었다. -59 bp 이상의 부위가 결손된 pUX35의 경우에는 전혀 xylA 유전자의 발현이 일어나지 않았다. 이러한 결과로 볼때 xylA 프로모터내의 조절부위는 -165 bp에서 -59 bp 사이에 존재하는 것으로 추정되었다.

• Applied Biological Chemistry
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• 제38권1호
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• pp.7-12
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• 1995

본 연구는 lacZ' 유전자의 promoter 개발을 위하여 착수하였다. lacZ' 유전자의 heterologous promoter I과 II를 효모 염색체의 Bam HI DNA 단편에서 분리하였다. Promoter I의 크기는 2.5 Kb 정도이고 ${\beta}-galactosidase$ 활성은 124.6 U/mg protein이었으며 promoter II의 크기와 효소활성은 4.0 Kb와 168.8 U/mg이었다. 형질 전환체에서의 YEp plasmid 안정성은 52.7%에서 67.4% 정도였다. YEp plasmid로부터 YIp plasmid를 재조합하였으며 이 YIp plasmid는 대장균에서나 효모에서도 발현되었다. 효모로부터 분리한 promoter I과 II는 재조합된 YEp와 YIp plasmid의 promoter로서 이용 가능하였다.