• Journal of Microbiology and Biotechnology
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• v.12 no.5
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• pp.841-843
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• 2002

Pfu DNA polymerase from Pyrococcus furiosus was expressed in the E. coli periplasm, and the fully active polymerase was partially purified by applying osmotic shock, ammonium sulfate precipitation, and heat treatment. This method represents a new way of expressing and purifying functional Pfu DNA polymerase without the use of chromatography.

• 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.

• KSBB Journal
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• v.31 no.1
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• pp.27-32
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• 2016

Several CoA transferases from Clostridium beijerinckii, C. perfringens and Klebsiella pneumoniae were examined for biosynthesis of lactate-containing polyhydroxyalkanoates (PHAs) in recombinant Escherichia coli XL1-Blue strain. The CB3819 gene and the CB4543 gene from C. beijerinckii, the pct gene from C. perfringens and the pct gene from K. pneumoniae, which encodes putative CoA transferase gene, respectively, was co-expressed with the Pseudomonas sp. MBEL 6-19 phaC1437 gene encoding engineered Pseudomonas sp. MBEL 6-19 PHA synthase 1 ($PhaC1_{Ps6-19}$) to examine its activity for the construction of key metabolic pathway to produce poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)]. The recombinant E. coli XL1-Blue expressing the phaC1437 gene and CB3819 gene synthesized poly(3-hydroxybutyrate) [P(3HB)] homopolymer to the P(3HB) content of 60.5 wt% when it was cultured in a chemically defined medium containing 20 g/L of glucose and 2 g/L of sodium 3-hydroxybutyrate. Expression of the phaC1437 gene and CB4543 gene in recombinant E. coli XL1-Blue also produced P(3HB) homopolymer to the P(3HB) content of 51.2 wt% in the same culture condition. Expression of the phaC1437 gene and the K. pneumoniae pct gene in recombinant E. coli XL1-Blue could not result in the production of PHAs in the same culture condition. However, the recombinant E. coli XL1-Blue expressing the phaC1437 gene and the C. perfringens gene could produce poly(3-hydroxybutyrate-co-lactate [P(86.4mol%3HB-co-13.7 mol%LA) up to the PHA content of 10.6 wt% in the same culture condition. Newly examined CoA transfereases in this study may be useful for the construction of engineered E. coli strains to produce PHA containing novel monomer such lactate.

• Biomedical Science Letters
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• v.8 no.3
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• pp.107-113
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• 2002

Enterohemorrhagic Escherichia coli (EHEC) strains of serotype O157:H7 have been shown to colonize the intestinal epithelial cell by the attaching and effacing (AE) mechanism. The AE lesion is mediated by an intimin, of which production and expression are controlled by a 3-Kb eaeA gene located EHEC chromosomal DNA. If the eaeA gene is mutated, EHEC O157:H7 strains lose capacity of adhesion to intestinal epithelial cells. In this study, a 891 bp of the 3'-end region of a gamma intimin was amplified by polymerase chain reaction (PCR). The PCR product was inserted into pSTBlue-1 cloning vector and transformed into DE3 (BL21) competent cell. After plasmid mini-preparation and restriction enzyme digestion of eaeA/891-pSTBlue-1 vector, target eaeA gene was re-inserted into pET-28a expression vector and was transformed. Then the expression of recombinant eaeA/891 (891 bp) gene was induced by isopropyl-$\beta$-D-thiogalactopyranoside (IPTG). The expression of the 40-KDa recombinant protein was identified in SDS-PAGE and confirmed by immunoblotting using the His.Tag$^{\circledR}$ and T$_{7}$.Tag$^{\circledR}$ monoclonal antibody. This recombinant protein expressed by eaeA gene could be applied in further studies on the mechanisms of E. coli O157:H7 infection and the development of recombinant vaccine.

• Korean Journal of Veterinary Research
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• v.46 no.1
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• pp.13-19
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• 2006

Generally known psittacosis or ornithosis is a disease of birds caused by the bacterium Chlamydia psittaci. Humans are accidential hosts and are most commonly infected from avian sources. It raises hepatitis or neurosis. As major outer membrane protein (MOMP) of Chlamydia psittaci has been known to play a role in the avoidance of host immune defenses, research on developing a Chlamydia vaccine has focused on the MOMP. In this study, the gene encoding the major outer membrane protein (MOMP) of the Chlamydia psittaci strain 6BC was cloned and expressed in Escherichia coli strain M-15. The recombinant DNA was cloned by fusion prokaryotic expression vector pQE30-GFPII. Expression of the recombinant protein was performed in E. coli and was induced by IPTG. The size of expressed recombinant protein is 74.220 kDa (MOMP, 43.260 kDa; GFP expression region, 30 kDa; $6{\times}His$ tag, 960Da). This protein was purified by using his-tagging-inclusion body. Recombinant protein was reconfirmed through ELISA test and western blot with antibody against pQE30-GFPII. It will be useful antibody development.

• Korean Journal of Microbiology
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• v.30 no.6
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• pp.539-545
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• 1992

Recombinant plasmid pPLac15 determined both phosphoenolpyruvate-dependent phosphotransferase uptake of lactose and phospho-$\beta$-galactosidase (Moon et al., 1989). A restriction mapping of the pPLac15 was compiled with several restriction enzymes and a seriese of sub clones into pUC18 was constructed. From an analysis of the proteins produced by Escherichia coli cells of transformants containing each of the recombinant subclone plasmids, it was found that the gene for phospho-$\beta$-galactosidase in pUCI8 was expressed about 1.8-folds in E. coli.

• Microbiology and Biotechnology Letters
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• v.23 no.6
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• pp.652-658
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• 1995

We attempted simultaneous expression of genes coding for endoglucanase and $\beta $-glucosidase from Pseudomonas sp. by using a synthetic two-cistron svstem in Escherichia coli and Saccharomyces cerevisiae. Two-cistron system, 5'--tac promoter-endoglucanase gene--$\beta $-glucosidase gene-- 3', 5'-tac promoter--$\beta $-glucosidase gene--endoglucanase gene--3' and 5'-tac promoter--endoglucanase gene--SD sequence--$\beta $-glucosidase gene--3, were constructed, and expressed in E. coli and S. cerevisiae. The E. coli and S. cerevisiae contained two-cistron system produced simultaneously endoglucanase and $\beta $-glucosidase. The recombinant genes contained the bacterial signal peptide sequence produced low level of endoglucanase and $\beta $-glucosidase in S. cerevisiae transformants: Approximately above 44% of two enzymes was localized in the intracellular fraction. The production of endoglucanase and $\beta $-glucosidase in veast was not repressed in the presence of glucose or cellobiose. The veast strain contained recombinant DNA with two genes hydrolyzed carboxvmethyl cellulose, and these endoglucanase and $\beta $-glucosidase degraded CMC synergistically to glucose, cellobiose and oligosaccharide. This result suggests the possibility of the direct bioconversion of cellulose to ethanol by the recombinant yeast.

• Journal of Microbiology and Biotechnology
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• v.13 no.5
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• pp.660-665
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• 2003

High-level expression of Thermus caldophilus GK24 alkaline phosphatase (Tca APase) was achieved in Escherichia coli using the pET-based expression plasmids, pEAP1 and pEAP2. In the case of plasmid pEAP2, the signal peptide region of Tca APase was replaced by the PelB leader peptide of expression vector pET-22b(+). Furthermore, the expression level was somewhat higher than that of plasmid pEAPl. A rapid purification procedure of Tca APase overproduced in E. coli was developed which involved heating to denature E. coli proteins followed by HiTrap Heparin HP column chromatography. Optimal temperature and pH and $Mg^{2+}$ dependence of the recombinant Tca APase were similar to those of native enzyme isolated from T. caldophilus GK24.

• Microbiology and Biotechnology Letters
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• v.23 no.4
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• pp.446-453
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• 1995

$\alpha $-Arabinofuranosidase was produced by E. coli HB101 haboring the recombinant plasmid pKMG11 which contained the arfI gene of Bacillus stearothermophilus. The maximum production of the enzyme was observed when E. coli HB101 cells were grown at 37$\circ$C for 20 hours in the medium containing 0.5% arabinose, 1.0% tryptone, 0.5% yeast extract, and 1% NaCl. The $\ALPHA $-arabinofuranosidase produced was purified to homogeneity using a combination of 20-50% ammonium sulfate precipitation, DEAE-Sepharose CL-6B ion exchange column chromatography and Sepharose 6B-100 gel filtration. The purified enzyme was most active at 55$\circ$C and pH 6.5. The K$_{m}$ and V$_{max}$ values of the enzyme on $\rho $-nitrophenyl-$\alpha $-arabinofuranoside was determined to be 2.99 mM and 0.43 $\mu $mole/min (319.74 $\mu $mole/min/mg), respectively. The pI value was 4.5. The molecular weight of the native protein was estimated to be 289 kDa. The SDS-polyacrylamide gel clectrophoresis analysis suggested that the functional protein was a trimer of the 108 kDa identical subunits. The N-terminal amino acid sequence of the a-arabinofuranosidase was identified as X-Ser-Thr-Ala-Pro-Arg( \ulcorner )-Ala-Thr-Met-Val-Ile-Asp-X-Ala-Phe.

• Microbiology and Biotechnology Letters
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• v.15 no.5
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• pp.346-351
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• 1987

A cellulase gene of Clostridium themocellum was transferred to Escherichia coli by molecular cloning with pBR322. The gene was carried in a Hind III digested DNA sequence of about 1.8 kb. This Rind III fragment expressed activities on carboxymethyl cellulose (CMC) and on filter gaper in E. coli. The expression of clostridial cellulase gene in E. coli was studied and compared with the pro-ducts of cellulase genes in C. themocellum.