• Journal of Microbiology and Biotechnology
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• 제7권1호
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• pp.8-12
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• 1997

In order to overproduce D-xylose isomerase, the Escherichia coli D-xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) gene (xylA) was fused to ${\lambda}P_{L}$ promoter. The promoterless xylA gene containing the ribosome binding site and coding region for D-xylose isomerase was cloned into a site 0.3 kb downstream from the ${\lambda}P_{L}$ promoter on a high copy number plasmid. An octameric XbaI linker containing TAG amber codon was inserted between 33rd codon of ${\lambda}N$ and the promoterless xylA gene. The resulting recombinant plasmid (designated as pPX152) was transformed into E. coli M5248 carrying a single copy of the temperature sensitive ${\lambda}cI857$ gene on its chromosomal DNA. When temperature-induced, the transformants produced 15 times as much D-xylose isomerase as that of D-xylose-induced parent strain. The amount of overproduced D-xylose isomerase was found to be about 60% of total protein in cell-free extracts.

• Journal of Microbiology and Biotechnology
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• 제18권4호
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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.

• Applied Biological Chemistry
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• 제31권3호
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• pp.219-225
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• 1988

Avian Sarcoma Virus의 plasmid DNA중(中)의 역 이사효소의 유전자(遺傳子)를 온도의존성(溫度依存性) 발현(發現) vector인 pPL-lambda에 cloning하여 온도(溫度)에 민감한 phage ${\lambda}$의 repressor인 cI857 gene을 갖고 있는 bacteriophage lysogen인 N4830에 transformation시켰다. transformant를 pL promoter의 발현(發現)을 억제(抑制)하는 저온(低溫)$(28^{\circ}C)$에서 배양(培養)시킨 뒤, 이 repressor를 억제(抑制)하여 transcription을 촉진(促進)하게 하는 고온(高溫)$(42^{\circ}C)$에서 배양(培養)시킨 다음 균체(菌體)를 회수(回收)하여 RNA를 추출(抽出)하고 분석(分析)을 한 결과(結果) 도입(導入)된 역전사 효소 유전자(遺傳子)의 전사(轉寫)가 고온(高溫)에서 증대(增大)되었다.

• Journal of Microbiology and Biotechnology
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• 제5권1호
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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.

• 미생물학회지
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• 제26권4호
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• pp.291-297
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• 1988

신호배열 돌연변이인 rbsB 103는 전구체 리보스 결합단백질을 세포질내에 축적시키고, rbsB 106 복귀유전자는 이 전구체를 숙성 가능하게 하여 페리플라슴으로 수송되게 한다.(Iida el at., 1985, Park, el at., 1988). 본고에서는 rbsB 유전자의 세 allele, rbsB, rbsB 103와 rbsB 106를 이들이 코딩하는 단백질을 대량생산 하그l자 람다 P 프로모터 조절하에 클론하고 나아가서 다섯종의 단백질을 순수정제하였다.. 전구체 단백질의 pI는 8.0, 숙성단백질의 $P_{L}$는 7.5임을 밝혔다. 순수정제된 단백질의 아마노 말단의 아미노산 배열을 결정하여 DNA 염기서열로부터 밝혀진 아미노산 변화를 확인하였다.

• 한국미생물·생명공학회지
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• 제19권5호
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• pp.433-438
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• 1991

Phenylalanine 고생산용 plasmid pSY130-14는 $\lambda$-phage 유래 온도감수성을 가지고 있으므로 $cI_{857}$ repressor와 PL과 PR을 온도를 올려 phenylalanine의 생산을 유도한다. pSY130-14를 갖는 E.coli AT2471은 kanamycin 무첨가, $38.5^{\circ}C$, 48시간에서 약 30%로 plasmid가 없어지며 첨가에서 안정성이 떨어졌다가 배양시간과 더불어 올라갔는데, 이것은 생육에 피요한 kanamycin gene과 ori만이 남는 것으로 생각된다.

• 한국독성학회:학술대회논문집
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• 한국독성학회 2000년도 국제심포지움 및 추계학술대회
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• pp.45-67
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• 2000

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a mutagenic and carcinogenic heterocyclic amino found in cooked meat. The in vivo mutagenicity and hepatocarcinogenicity of MeIQx were examined in mice harboring the lacZ mutation reporter gene ($Muta^{TM}$ Mice) and bitransgenic mice over-expressing the c-myc oncogene. C57B1/$\lambda$lacZ and bitransgenic c-myc (albumin promoter)/$\lambda$lacZ mice were bred and weaned onto an AIN-76 based diet containing $0.06\%$ (w/w) MeIQx or onto control diet. After 30 weeks on diet, only male bitransgenic mice on MeIQx developed hepatocellular carcinoma ($100\%$ incidence) indicating that there was synergism between c-myc over-expression and MeIQx. By 40 weeks, hepatic tumor incidence was $100\%$ ($17\%$) and $44\%$ ($0\%$) in male c-myc/$\lambda$lacZ and C57B1/$\lambda$lacZ mice given MeIQx (or control) diet, respectively, indicating that either MeIQx or c-myc over-expression alone eventually induced hepatic tumors. At either time point, mutant frequency in the lacZ gene was at least 40-fold higher in MeIQx-treated mice than in control mice of either strain. These findings suggest that MeIQx-induced hepatocarcinogenesis is associated with MeIQx-induced mutations. Elevated mutant frequency in MeIQx-treated mice also occurred concomitant with the formation of MeIQx-guanine adducts as detected by the $^{32}P$-postlabeling assay. Irrespective of strain or diet, sequence analysis of the lacZ mutants from male mouse liver showed that the principal sequence alteration was a single guanine-base substitution. Adenine mutations, however, were detected only in animals on control diet. MeIQx-fed mice harboring the c-myc oncogene showed a l.4-2.6-fold higher mutant frequency in the lacZ gene than mice not carrying the transgene. Although there was a trend toward higher adduct levels in c-myc mice, MeIQx-DNA adduct levels were not significantly different between c-myc/$\lambda$lacZ and C57B1/$\lambda$lacZ mice after 30 weeks on diet. Thus, it appeared that factors in addition to MeIQx-DNA adduct levels, such as the enhance rate of proliferation associated with c-myc over-expression, may have accounted for a higher mutant frequency in c-myc mice. In the control diet groups, the lacZ mutant frequency was significantly higher in c-myc/$\lambda$lacZ mice than in 057B1/$\lambda$1acZ mice. The findings are consistent with the notion that c-myc over-expression is associated with an increase in mutagenesis. The mechanism for the synergistic effects of c-myc over-expression on MeIQx hepatocarcinogenicity appears to involve an enhancement of MeIQx-induced mutations.

• 미생물학회지
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• 제27권3호
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• pp.169-175
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• 1989

포유동물 세포내에서 간염 바이러스의 내면항원의 발현과 전위내면 항원(precore) 부위의 역할을 규명하기 위하여 고등동물세포 발현용 벡터에 전위내면항원 부위를 갖거나 또는 갖지 않는 내면항원 유전자를 클로닝 하여 COS 세포내에서의 발현을 조사하였다. 전위내면항원 부위를 포함한 내면항원 유전자를 갖는 플라스미드로감염시킨 COS 세포는 항원들이 세포추출물과 배양액에서 검출되었다. 분비된 항원의 증가율은 감염후 2일과 3일 사이가 가장 높았고, 부분결실된 제조합 플라스미드 중 내면항원의 ATG codon에서 180bp 떨어진 것이 가장 발현이 잘 되었다. 전위내면항원을 갖지 않거나 하나의 염기가 첨가되어 변형된 전위내면항원을 갖는 제조합 플라스미드의 경우 항원들이 세포 추출물에서만 검출되었다. 이러한 사실은 전이내면항원 부위가 HBe 항원의 분비에 관여 한다는 경우 항원들이 세포 그러나 대장균이나 효모 세포의 경우는 전위내면항원의 존재와 상관없이 항상 세포추출물에서만 존재하는 것으로 보아 이들 세포의 경우에서는 전위내면항원 부위가 HBe 항원의 분비에 영향을 줄 수 없음을 의미한다.