• 한국미생물·생명공학회지
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• 제36권4호
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• pp.260-267
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• 2008

단백질의 산업적 생산을 위해 발현벡터의 선정이 중요하지만 이용 가능한 프로모터가 극히 제한적이며 많은 경우 과발현되는 특성과 함께 불용성 응집체가 형성되는 단점을 지닌다. 따라서 다양한 생물로부터 유래된 잠재성이 큰 유전자원(metagenome)에서의 프로모터 발굴과 한정된 숙주를 해결하려는 노력이 요구된다. 선행연구에서 발굴한 metagenome 유래의 항시발현 프로모터를 이용해 대장균의 일반적인 배양조건에서 세포생리에 영향이 적은 신규 항시발현 벡터를 제작하였다. 이를 위해 예측된 프로모터 서열과 MCS를 포함하는 합성 primer를 제작한 후 PCR로 증폭해 발현벡터를 구성한 후, 프로모터 구동여부와 단백질 발현양상 등을 관찰하였다. 인위적으로 도입된 MCS에 GFP, esterase, $\beta$-glucosidase를 클로닝해 단백질 발현양과 가용성을 분석한 결과, 안정적으로 전체단백질의 $2{\sim}3%$ 정도로 발현되며 80% 이상의 높은 가용성을 지닌 단백질의 발현이 유도되는 것으로 확인되었다. 이와 같은 결과는 잠재적인 생물자원의 보고로서 metagenome의 활용가능성을 제시하고 있다. 따라서 다양한 숙주에서 작동하는 프로모터의 발굴 및 발현벡터의 제작을 시도할 경우 단백질의 생산이나 대사공학에 의한 균주개량에 유용하게 활용할 수 있을 것이다.

• Journal of Microbiology and Biotechnology
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• 제20권11호
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• pp.1563-1570
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• 2010

To construct a highly sensitive detection system for endocrine disruptors (EDs), we have compared the activity of promoters with the n-alkane-inducible cytochrome P450 gene (ALK1), isocitrate lyase gene (ICL1), ribosomal protein S7 gene (RPS7), and the translation elongation factor-1${\alpha}$ gene (TEF1) for the heterologous gene in Yarrowia lipolytica. The promoters were introduced into the upstream of the lacZ or hERa reporter genes, respectively, and the activity was evaluated by ${\beta}$-galactosidase assay for lacZ and Western blot analysis for hER${\alpha}$. The expression analysis revealed that the ALK1 and ICL1 promoters were induced by n-decane and by EtOH, respectively. The constitutive promoter of RPS7 and TEF1 showed mostly a high level of expression in the presence of glucose and glycerol, respectively. In particular, the TEF1 promoter showed the highest ${\beta}$-galactosidase activity and a significant signal by Western blotting with the anti-estrogen receptor, compared with the other promoters. Moreover, the detection system was constructed with promoters linked to the upstream of the expression vector for the hER${\alpha}$ gene transformed into the Y. lipolytica with a chromosome-integrated lacZ reporter gene under the control of estrogen response elements (EREs). It was indicated that a combination of pTEF1p-hER${\alpha}$ and CXAU1-2XERE was the most effective system for the $E_2$-dependent induction of the ${\beta}$-galactosidase activity. This system showed the highest ${\beta}$-galactosidase activity at $10^{-6}\;M\;E_2$, and the activity could be detected at even the concentration of $10^{-10}\;M\;E_2$. As a result, we have constructed a strongly sensitive detection system with Y. lipolitica to evaluate recognized/suspected ED chemicals, such as natural/synthetic hormones, pesticides, and commercial chemicals. The results demonstrate the utility, sensitivity, and reproducibility of the system for identifying and characterizing environmental estrogens.

• 한국환경성돌연변이발암원학회:학술대회논문집
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• 한국환경성돌연변이발암원학회 2002년도 Molecular and Cellular Response to Toxic Substances
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• pp.90-90
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• 2002

• Journal of Pharmaceutical Investigation
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• 제15권4호
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• pp.198-200
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• 1985

• Journal of Pharmaceutical Investigation
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• 제15권4호
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• pp.231-245
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• 1985

• 한국유전체학회:학술대회논문집
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• 한국유전체학회 2003년도 The 12th Korea Genome Conference
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• pp.41.1-41.1
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• 2003

• BMB Reports
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• 제28권4호
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• pp.368-373
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• 1995

The upstream regulatory region (URR) of bovine papillomavirus type 1 (BPV) contains promoters and a conditional transcriptional enhancer that is trans-activated by the viral E2 protein. After deleting the 5' and 3' ends of BPV URR, BamHI linkers were inserted into several positions of BPV URR without causing an addition or a deletion of URR sequences. Most linker scanning mutations did not show any effects on the transcription of P7940 and P89 promoters in BPV URR. However, several mutants showed reduced transcriptional activities. Based on our results we found that the AP-2 and Sp1 binding sites were important for basal level transcription of BPV URR in the absence of the E2 protein and that the CTF/NF-1 site is dispensable for E2 transactivation of BPV URR transcription.

• BMB Reports
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• 제30권5호
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• pp.320-325
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• 1997

Thioredoxin is a multi-functional protein which is ubiquitous in microorganisms, animals and plants. Previously, expression of the E. coli thioredoxin gene was found to be negatively regulated by cAMP. In the present study, the effect of cAMP on two separate promoters of the E. coli thioredoxin gene was investigated. Cyclic AMP had a repressible effect on P1 and P1P2 promoter activity of the constructs. This effect was also observed in the cya strain. The P2 promoter construct gave very high -galactosidase activity, and its expression was not affected by exogenous cAMP. It was assumed that a cis-acting negative element, probably the cAMP-CRP binding site, might have been deleted in the P1 promoter construct. Repression of the thioredoxin gene expression by cAMP appeared to be independent of ppGpp.

• Asian-Australasian Journal of Animal Sciences
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• 제20권6호
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• pp.1007-1014
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• 2007

Acceptance of antibiotic growth promoters (AGP) in agricultural animal production is rapidly disappearing. Both government regulations and consumer preference are driving this change. Producers in any country that seek export markets will be forced to give up AGP if they are to sell to the EU and many other markets. This report will first review the history of AGP use in the animal industry and the concerns about development of antimicrobial resistance. A description of the development and structure of the gut and how it is affected by AGP administration will conclude with results of studies to replace AGP with antimicrobial organic acids.

• Asian-Australasian Journal of Animal Sciences
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• 제19권10호
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• pp.1490-1495
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• 2006

The purpose of this research was to develop an optimum composition model for a new synbiotic fermented dairy product with high probiotic cell counts, and to experimentally verify this model. The optimum composition model indicated the growth promoter ratio that could provide the highest growth rate for probiotics in this fermented product. Different levels of growth promoters were first blended with milk to improve the growth rates of probiotics, and the optimum composition model was determined. The probiotic viabilities and chemical properties were analyzed for the samples made using the optimal formula. The optimal combination of the growth promoters for the synbiotic fermented milk product was 1.12% peptides, 3% fructooligosaccharides (FOS), and 1.87% isomaltooligosaccharides (IMO). A product manufactured according to the formula of the optimum model was analyzed, showing that the model was effective in improving the viability of both Lactobacillus spp. and Bifidobacterium spp.