• The Korean Journal of Zoology
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• v.33 no.3
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• pp.303-309
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• 1990

In order to have a handle on the availability of eukarvotic peptide hormone precursors, a cDNA encoding angler fish prepro-SRIF I was manipulated so that it can be produced in large quantity from heterologous E. coli cells. Using T7 overexpression system, fusion constructs between the T7 phage coat protein Sl0 and the prepro-SRIF were made and modified as desired. From the host E. coli strain, BL21 DE3, harboring these plasmid constructs, three different SRIF related polypeptides were expressed in large amount and characterized. The results confirm the exact construction and authenticity of the overexpressed proteins from E. coli cells. The importance of this heterologous overexpression in hard to get peptide hormone precursors as well as the suitability of the target peptide hormone SRIF for this approach are discussed.

• Journal of Microbiology and Biotechnology
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• v.29 no.3
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• pp.401-409
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• 2019

Heat-resistant microbial hosts are required for bioprocess development using high cell density cultivations at the industrial scale. We report that the thermotolerance of Escherichia coli can be enhanced by overexpressing ybeD, which was known to encode a hypothetical protein of unknown function. In the wild-type E. coli BL21(DE3), ybeD transcription level increased over five-fold when temperature was increased from $37^{\circ}C$ to either $42^{\circ}C$ or $46^{\circ}C$. To study the function of ybeD, a deletion strain and an overexpression strain were constructed. At $46^{\circ}C$, in comparison to the wild type, the ybeD-deletion reduced cell growth half-fold, and the ybeD-overexpression promoted cell growth over two-fold. The growth enhancement by ybeD-overexpression was much more pronounced at $46^{\circ}C$ than $37^{\circ}C$. The ybeD-overexpression was also effective in other E. coli strains of MG1655, W3110, DH10B, and BW25113. These findings reveal that ybeD gene plays an important role in enduring high-temperature stress, and that ybeD-overexpression can be a prospective strategy to develop thermotolerant microbial hosts.

• Journal of Microbiology and Biotechnology
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• v.19 no.1
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• pp.72-77
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• 2009

Here, we demonstrate that the overexpression of the GroELS chaperone system, which assists the folding of intracellular proteins and prevents aggregation of its biological targets, can enhance the thermotolerance of Escherichia coli strains and facilitate the production of recombinant protein under thermal stress. The overexpression of GroELS led to an about 2-fold higher growth rate of E. coli XL-1 blue than control at $45^{\circ}C$ and induced the growth of the strain even at $50^{\circ}C$, although the growth was not sustained in the second-round culture. The effect of GroELS overexpression was also effective on other E. coli strains such as JM109, $DH5{\alpha}$, and BL21. Finally, we have shown that coexpression of GroELS allows us to produce recombinant protein even at $50^{\circ}C$, a temperature at which the protein production based on E. coli is not efficient. This study indicates that the employment of the GroELS overexpression system can expand the range of environmental conditions for E. coli.

• KSBB Journal
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• v.19 no.4
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• pp.331-334
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• 2004

In vivo characterization of FadB homologous enzymes including PaaG, YdbU and YgfG for medium-chain-length (MCL) polyhydroxyalkanoate (PHA) biosynthesis was carried out in fadB mutant Escherichia coli. Previously, it was reported that amplification of FadB homologous enzymes such as PaaG and YdbU in fadB mutant E. coli resulted in enhanced biosynthesis of MCL-PHA by greater than two fold compared with control strain. In this study, we constructed paaG fadB double mutant E. coli WB114 and ydbU fadB double mutant E. coli WB115 to investigate the roles of PaaG and YdbU in biosynthesis of MCL-PHA. Inactivation of paaG and ydbU genes in fadB mutant E. coli harboring Pseudomonas sp. 61-3 phaC2 gene reduced the MCL-PHA production to 0.16 and 0.16 PHA g/L, respectively from 2 g/L of sodium decanoate, which are much lower than 0.43 PHA g/L obtained with fadB mutant E. coli WB101 harboring the phaC2 gene. Also, we identified new FadB homologous enzyme YgfG, and examined its roles by overexpression of ygfG and construction of ygfG fadB double mutant E. coli WB113.

• Journal of Microbiology and Biotechnology
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• v.8 no.1
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• pp.92-95
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• 1998

Overproduction of Escherichia coli thiol peroxidase in the periplasmic space was achieved by locating the appropriate gene on a downstream region of the strong T7 promoter. E. coli strain BL21 carrying the recombinant plasmid pSK-TPX was induced by IPTG, lysed, and analyzed by SDS-polyacrylamide gel electrophoresis. A large amount of the overexpressed thiol peroxidase was located in the periplasmic space. A homogeneous thiol peroxidase was obtained from E. coli osmotic shock fluid by simple one-step gel permeation chromatography.

• Journal of Life Science
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• v.9 no.2
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• pp.62-66
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• 1999

To overexpress E. coli ADA in host strain E. coli M15, the expression plasmid pQEADD was constructed. To analyze the expression characteristics of ADA, a time course of the expression was first examined. The protein was not detected in no inducted in no induction samples. After the addition of IPTG, a band corresponding to the expected size of ADA was appeared. Its molecular weight was about 36,000 dalton. Maximum expression level was revealed when the cell cultured for 3∼4hrs after induction. This result indicated that the efficient expression of add can be achieved by induction at early logarithmic phase. The effect of different IPTG concentration on the degree of ADA expression was investigated. The expression levels of add were not largely affected by IPTG concentration. Location of overexpression ADA was checked out. A protein band corresponding to the ADA was seen in only crude extract B(insoluble protein). This result suggests that ADA is in E. coli M15. The molecular weight of ADA estimated by SDS-PAGE was approximately 36,000 Da.

• Korean Journal of Microbiology
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• v.30 no.2
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• pp.141-148
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• 1992

In order to overexpress bacteriophage PRD1 DNA polymerase in E. coli cells, the 2 kb HaeII fragment was isolated from phage genomic DNA. This fragment was then cloned into pEMBL/sup ex/ 3-expression vector. A specific 57bp deletion was performed by using uracil containing ss DNA and oligonucleotide spanning each region to remove an unwanted non-coding region. After this deletion, the PRD1 DNA polymerase gene is totally under the control of the vector promoter and SD sequence. Upon heat induction, a protein with an apparent size of 68 kdal was overexpressed as an active PRD1 DNA polymerase. The expression of PRD1 DNA polymerase was about 1% of total E. coli protein.

• Journal of Microbiology and Biotechnology
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• v.14 no.4
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• pp.777-782
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• 2004

The arylsulfatase gene (astA, 984 bp ORF) from the P. carrageenovora genome was amplified by PCR and subcloned into the pET21a vector. When the constructed plasmid pAST-A1 (6.4 kb) was introduced into E. coli BL21(DE3), the transformant on the LB plate containing IPTG showed a hydrolyzing activity for 4-methylumbelliferyl sulfate and p-nitrophenyl sulfate. The highest arylsulfatase activity (2.1 unit/ml) was obtained at 10 mM IPTG. Most arylsulfatase activity was found in the cell lysate, whereas no significant activity was detected in the culture supernatant. The molecular weight of the recombinant enzyme was estimated to be 33.1 kDa by SDS-PAGE. After the reaction of agar with arylsulfatase for 12 h at $40^{\circ}C$, the gel strength of the agar increased by 2-fold, and 73% of the sulfate in the agar had been removed. This result suggests that arylsulfatase expressed in E. coli could be useful in the production of electrophoretic grade agarose.

• Journal of Microbiology and Biotechnology
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• v.16 no.9
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• pp.1448-1452
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• 2006

The effects of amplifying the gluconeogenic phosphoenolpyruvate carboxykinase of Escherichia coli ($pck_{Ec}$) on succinic acid production in E. coli were examined under anaerobic condition. No significant increase in succinic acid production was observed in E. coli overexpressing the $pck_{Ec}$ gene without supplementing $NaHCO_{3}$ or $MgCO_{3}$. On the other hand, succinic acid production was enhanced as the $NaHCO_{3}$ concentration was increased. When 20 g/l of $NaHCO_{3}$ was added, succinic acid production in recombinant E. coli overexpressing PCK was 2.2-fold higher than that observed in the wild-type strain. It was concluded that the gluconeogenic $pck_{Ec}$ overexpression enabled E. coli to enhance succinic acid production only under the high bicarbonate supplementation condition.

• Journal of Microbiology and Biotechnology
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• v.24 no.4
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• pp.568-576
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• 2014

TIM-1 (also known as KIM-1 and HAVcr-1) is a type I transmembrane glycoprotein member of the TIM family that may play important roles in innate and adaptive immune responses. The overexpression of proteins associated with membrane proteins is a major obstacle to overcome in studies of membrane protein structures and functions. In this study, we successfully coupled the overexpression of the TIM-1 protein with a C-terminal enhanced green fluorescent protein (GFP) tag in Escherichia coli. To the best of our knowledge, this report is the first to describe the overexpression of human TIM-1 in E. coli. The purified TIM-1-EGFP fusion protein recognized and bound directly to apoptotic cells and did not to bind to viable cells. Furthermore, we confirmed that the interactions of TIM-1-EGFP with apoptotic cells were blocked by TIM-1-Fc fusion proteins. This fusion protein represents a readily obtainable source of biologically active TIM-1 that may prove useful in future studies of human TIM-1.