• Microbiology and Biotechnology Letters
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• v.20 no.2
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• pp.122-128
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• 1992

Runaway replication plasmid pSY35AT was used for the production of $cI_{857}$ repressor protein. E, coli MC1065 having plasmid pSY35AT was shifted from $30^{\circ}C$ to $37^{\circ}C$ $cI_{857}$ repressor protein produced was purified by a modification of the purification method of wild type cI repressor. The concentration of purified $cI_{857}$ repressor protein was 0.11 mg/ml. The binding assay of $cI_{857}$ repressor and right promoter - right operater ($P_RO_R$) labeled with $^3H-CTP$ was done. Relative activity of purified cIx5, repressor was higher about 23 times than that of cell free extract. A higher value of the temperature in the binding assay led to a lower value of the binding activity.

• Microbiology and Biotechnology Letters
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• v.19 no.1
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• pp.45-51
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• 1991

Inserting the $cI_{857}$ structural gene in the downstream of the tac promoter for the overproduction of $cI_{857}$ repressor protein was studied. DNA fragment containing $cI_{857}$ ; repressor gene was amplified by using plasmid pUC12, and partially digested with HphI. Only the $cI_{857}$ structural gene isolated was inserted in the downstream of the tac promoter. Plasmid pDR540- $cI_{857}$ having the tuc promoter-$cI_{857}$ structural gene insert could be isolated by the immunity of cells resistant at $30^{\circ}C$ and cell lysis at $42^{\circ}C$ to $\lambda$ phage $cI_{90}$. The amount of $cI_{857}$ repressor as 17% of total cellular protein were produced by using general E. coli as well as $lacI^q$ JM103 having this plasmid.

• Microbiology and Biotechnology Letters
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• v.19 no.5
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• pp.433-438
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• 1991

The plasmid pSY130-14 for the high production of phenylalanine is a temperaturecontrollable expression vector composed of the $P_R$ and the $P_L$ promoter and a temperature sensitive repressor, $cI_{857}$ of bacteriophage lambda. Strain AT2471 harbouring plasmid pSY13O- 14 is induced the phenylalanine production by shifting up the incubation temperaure to $38.5^{\circ}C$. Plasmid stability of E. coli AT2471 harbouring pSY130-14 was very low, it was about 30% after 48 h cultivation at $38.5^{\circ}C$ without kanamycin. The plasmid disappeared immediately at $40^{\circ}C$ without kanamycin, and at $40^{\circ}C$ adding kanamycin, the plasmid stability decreased at the beginning, but rose with the extension of the culture time. For the improvement of plasmid stability, the plasmid obtaind was designated as pSY15O-1 by changing origin region (ori) pACYC 177 of pSY130-14 for ori pSC101. E. coli AT2471 harbouring pSY150-1 was stable at $38.5^{\circ}C$ without tetracycline, and the plasrnid stability was about 40% after 48 h cultivation at $40^{\circ}C$.

• Applied Biological Chemistry
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• v.31 no.3
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• pp.219-225
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• 1988

Reverse transcriptase gene of Avian sarcoma virus(ASV) was cloned with a thermoinducible expression vector, pPL-lambda. E. coli N4830 which carries temperature sensitive cI857 la mbda repressor, was transformed with this pPL-pol plasmid DNA. The RNA transcribed by those tranoformants was isolated and analyzed. It was shown that the inserted reverse transcriptase gene of ASV was transcribed at high-level when cells were grown at high temperature.

• Korean Journal of Microbiology
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• v.29 no.2
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• pp.80-84
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• 1991

We cloned and expressed hepatitis B viral core antigen (HBcAg) gene in E. coli using $P_{L}$ promoter system. For optimal expression of the gene, we undertook the studies on the effects of the distance between Shine-Dalgarno (SD) sequence and start codon, copy number of repressor gene, induction temperature, and the stability of the core antigen. The results demonstrated that the induction at 37.deg.C was more efficient than at 42.deg.C, and the 11 base pairs (bp) distance between SD sequence and start codon of HBcAg gene was more efficient than the 15 bp distance in E. coli. The copy number of cI857 repressor gene did not influence on the expression of HBcAg, and the expression level of HBcAg in mutant type (low protease activity) and wild type strains was almost the same. The produced core antigen appeared to be HBcAg not HBeAg judged by two different radioimmunoassat (RIA) kits. This result suggested that the antigen was stable in E. coli.i.

• Journal of Microbiology and Biotechnology
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• v.15 no.6
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• pp.1346-1352
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• 2005

We have developed a modified gene replacement method using PCR products containing short homologous sequences of 40- to 50-nt. The method required $\lambda$-Red recombination functions provided under the control of a temperature-sensitive CI857 repressor expressed from the $P_{lac}$ promoter in the presence of IPTG on an easily curable helper plasmid. The method promoted the targeted gene replacements in the Escherichia coli chromosome after shifting cultures of the recombinogenic host, which carries the helper plasmid, to $42^{\circ}C$ for 15 min. Since this method employs $\lambda$-Red recombination functions expressed from the easily curable helper plasmid, multiple rounds of gene replacements in the E. coli chromosome would be possible. The procedures described herein are expected to be widely used for metabolic engineering of E. coli and other bacteria.

• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.639-647
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• 2008

The heat-inducible expression vectors for Corynebacterium glutamicum and C. ammoniagenes were constructed by using the ${\lambda}O_L1$ and the cryptic promoters, CJ1 and CJ4 that express genes constitutively in C. ammoniagenes. Although the promoters were isolated from C. ammoniagenes, CJ1 and CJ4 were also active in C. glutamicum. To construct vectors, the $O_L1$ from the ${\lambda}P_L$ promoter was isolated and fused to the CJ1 and CJ4 promoters by recombinant PCR. The resulting artificial promoters, CJ1O and CJ4O, which have one ${\lambda}O_L1$, and CJ1OX2, which has two successive ${\lambda}O_L1$, were fused to the green fluorescent protein (GFP) gene followed by subcloning into pCES208. The expression of GFP in the corynebacteria harboring the vectors was regulated successfully by the temperature-sensitive cI857 repressor. Among them, C. ammoniagenes harboring plasmid pCJ1OX2G containing GFP fused to CJ1OX2 showed more GFP than the other ones and the expression was tightly regulated by the repressor. To construct the generally applicable expression vector using the plasmid pCJ1OX2G, the His-tag, enterokinase (EK) moiety, and the MCS were inserted in front of the GFP gene. Using the vector, the expression of pyrR from C. glutamicum was tried by temperature shift-up. The results indicated that the constructed vectors (pCeHEMG) can be successfully used in the expression and regulation of foreign genes in corynebacteria.

• Journal of Microbiology and Biotechnology
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• v.5 no.1
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• pp.14-17
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• 1995

Endoglucanase gene of Pseudomonas sp. LBC505 was previously cloned in pUC19 to yield plasmid pLCl. The Pseudomonas sp. LBC505 endoglucanase gene was subcloned in a temperature-regulated Es-cherichia coli expression vector, pAS1, containing the leftward promoter $P_L$ of bacteriophage lambda. The level of gene expression was controlled by the thermal inactivation of the heat-sensitive lambda cI857 repressor. Best yield of endoglucanase was obtained by lowering the incubation temperature to $37^{\circ}C$ after induction at $42^{\circ}C$ for 1h. Under these conditions enzyme production continued for about 5h at a gradually decreasing rate. Ecoli harboring recombinant plasmid pASC10 expressed 4.3 times as much CMCase activity as E.coli containing pLCl. To enhance the expression level of endogl, ucanase gene, we have also changed the presumptive Shine-Dalgamo sequence (AGAGGT) of the gene to consensus sequence (AGGAGGT) by site-directed mutagenesis. The genes mutated were subcloned in pASl resulting in the formation of recombinant plasmid pASS50. E.coli harboring the plasmid pASS50 expressed 6.2-fold higher levels of CMCase activity than that of E.coli harboring pLC1.

• Journal of Microbiology and Biotechnology
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• v.2 no.1
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• pp.26-34
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• 1992

The effect of induction temperature on fermentation parameters has been investigated extensively using Escherichia coli M5248[pNKM21], a producer of recombinant human interleukin-2 (rhIL-2). In this recombinant microorganism, the gene expression of rhIL-2 is regulated by the cI857 repressor and $P_L$ promoter system. The recombinant fermentation parameters studied in this work include the cell growth, protein synthesis, cell viability, plasmid stability, $\beta$-lactamase activity, and rhIL-2 productivity. Interrelationships of such fermentation parameters have been analyzed through a quantitative assessment of the experimental data set obtained at eight different culture conditions. While the expression of rhIL-2 gene was repressed at culture temperatures below $34^\circ{C}$ with little effect on other fermentation parameters, under the conditions of rhIL-2 production $>(36~44^\circ{C})$ the cell growth, plasmid stability, and $\beta$-lactamase activity were, as induction temperature was increased, more profoundly reduced. Although the rhIL-2 content in the insoluble protein fraction was maximum at $40^\circ{C}$, total rhIL-2 production in the culture volume was found to be highest at the induction temperature of $36^\circ{C}$. This was in contrast to the previously known optimum induction temperature of the P$_{L}$ promoter system $>(40~42^\circ{C})$.Explanations for such a discrepancy have been proposed based on a product formation kinetics, and their implications have been discussed in detail.l.