• Journal of Microbiology and Biotechnology
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• 제13권4호
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• pp.620-623
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• 2003

The delta sleep-inducing peptides (DSIP, Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu) is an important regulatory hormone, controlling hypothalamus and pituitary functions. In the current study, an expression system was designed for the rapid and economic expression oi recombinant DSIP for biophysical studies. Artificially synthesized oligonucleotides encoding DSIP were cloned into a pGEX-KG vector and expressed in E. coli (BL21). The recombinant GST-DSIP was then readily purified using a GST affinity column. To obtain intact DSIP from the GST-DSIP, thrombin cleavage and a CNBr reaction were successively carried out. The DSIP in the CNBr reaction mixture was subjected to RP-HPLC purification to yield 1.2 mg DSIP from a 1 liter culture of E. coli. Identification of the DSIP was peformed using MALDI-MS and an amino acid composition analysis.

• 한국자원식물학회지
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• 제30권1호
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• pp.1-7
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• 2017

본 연구는 항암 단백질로 알려진 lunasin peptide의 산업적 활용성을 높이고자 lunasin peptide를 생산할 수 있는 시스템을 개발하고, 생산된 lunasin peptide가 식물유래 lunasin peptide의 생리활성을 가지는지 chromatin binding 활성과 histone acetylation 억제활성을 통해 평가하였다. 그 결과 E. coli와 P. pastoris를활용하여 재조합 lunasin peptide를 생산했으며, 생산 된 재조합 lunasin 펩타이드가 식물유래 lunasin peptide의 chromatin binding 활성과 histone acetylation 억제활성을 나타냄을 확인할 수 있었다. 따라서, 본 실험 연구의결과를 토대로 lunasin 펩타이드의 대량생산이 진행된다면 천연물 유래 생리활성 물질로서 효과적이면서도 안전한 기능성 식품소재로의 산업적 활용이 가능할 것으로 기대된다.

• 한국미생물·생명공학회지
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• 제32권3호
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• pp.212-217
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• 2004

Glycosyl hydrolase family 26에 속하는 Bacillus subtilis WL-7 mannanase를 코드하는 유전자를 대장균에서 과잉 발현하였다. 아미노 말단의 signal peptide를 포함하거나 포함하지 않은 mannanase 유전자를 각각 pET24a(＋)에 도입하여 재조합 플라스미드 pETMAN과 pENS7를 제조하였다. 이들 플라스미드를 함유하는 Escherichia coli BL21(DE3)에서 mannanase를 발현시킨 결과 signal peptide가 제거된 mannanase유전자의 발현량이 매우 높았다. 그러나 배양온도 $37^{\circ}C$에서 pENS7를 함유한 재조합 대장균에서 과잉 발현된 mannanase는 대부분이 불활성 형태로 존재하였으며, 배양온도를 $31^{\circ}C$이하로 하였을 때 수용화 형태의 효소량이 증가하면서 효소활성이 높아졌다. IPTG에 의해 발현된 재조합 대장균의 균체파쇄 상등액 중에 존재하는 mannanase 활성은 배양온도 $25^{\circ}C$～28$^{\circ}C$에서 가장 높았으며, 전체 단백질량을 기준으로 볼 때는 배양온도 $31^{\circ}C$에서 비활성이 가장 높은 것으로 확인되었다.

• Molecules and Cells
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• 제26권6호
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• pp.616-620
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• 2008

Maintaining redox balance is one of the crucial requirements for a cell to endure stress from the outside. Dehydroascorbate reductase (DHAR; EC 1.8.5.1) plays an important role in the ascorbate-glutathione cycle; one of the major ROS scavenging systems in most known biological systems. A cDNA clone of the DHAR gene from Oryza sativa (OsDHAR) was isolated and overexpressed in Escherichia coli BL21 (DE3) strain from the pET-28a(+) expression vector. The OsDHAR transformed E. coli cells showed significantly higher DHAR activity and a lower level of ROS than the E. coli cells transformed by an empty pET-28a(+) vector. Also, the DHAR-overexpressing E. coli strain was more tolerant to oxidant- and heavy metal-mediated stress conditions than the control E. coli strain. The results suggest that the overexpressed rice DHAR gene effectively functions in a prokaryotic system and provide protection to various oxidative stresses.

• 대한미생물학회지
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• 제34권6호
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• pp.555-559
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• 1999

The 38 kDa protein of Mycobacterium tuberculosis, which was known previously as antigen 5, has been extensively used in the serodiagnosis of tuberculosis. In an attempt to develop and evaluate a serodiagnostic test using the antigen, we expressed the 38 kDa protein in BCG and its seroreactivity was compared to that expressed in Escherichia coli. The coding region of the 38 kDa protein was amplified by PCR, and the gene was cloned into a Mycobacterium-E. coli shuttle expression vector pYMC-his and pQE30 expression vector and expressed in BCG and E. coli, respectively. Both recombinant 38 kDa proteins showed strong seroreactivity against pooled serum from tuberculosis patients. There was no significant difference in seroreactivity between the two recombinant antigens in sera from the far advanced tuberculosis patients. However, of 25 tuberculosis patients graded as "minimal" by chest X-ray, 5 (20.0%) were seropositive by r38 kDa expressed in E. coli, while 8 (32.0%) by that expressed in BCG. Likewise, higher seroreactivity by r38 kDa expressed in BCG was found in sera from the moderately advanced tuberculosis. This study thus indicates that the recombinant 38 kDa expressed in BCG is more effective than that expressed in E. coli in detecting antibodies to the native 38 kDa protein of M. tuberculosis in sera from minimally affected tuberculosis patients.

• KSBB Journal
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• 제23권5호
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• pp.403-407
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• 2008

본 연구에서는 현재 산업적 응용이 활발하게 이루어지고 있고, 여러 장점을 지닌 효소인 Candida antarctica에서 유래된 lipase B (CalB)의 신속한 개질을 위해 취급이 용이한 E. coli를 이용하여 CalB 발현시스템을 구축하였다. E. coli 발현 시스템에서 효소활성을 지니지 못하는 내포체를 생성하는 단점을 지니고 있어, soluble한 형태의 CalB 생성을 위해 저온 발현이 가능한 pCold I vector와 단백질 접힘을 도와주는 chaperone을 사용하여 CalB를 발현하였다. Liu 등(17)은 E. coli Origami2와 B, 그리고 $DH5{\alpha}$를 실험한 결과, Origami 균주에서만 CalB에 의한 halo의 형성이 관찰되었으나, 동 연구에서는 실험한 3종의 균주와 5종의 chaperone plasmid중 Rosettagami와 $DH5{\alpha}$에서 groES/groEL chaperone이 CalB와 동시에 발현되면 soluble한 형태의 Cal B가 발현됨을 관찰할 수 있었다. 또한 신속한 CalB의 발현시스템을 구축하기 위해서는 유전자 조작의 용이성 및 안정성에서 우월한 $DH5{\alpha}$가 Rosettagami에 비해 soluble한 CalB의 발현에 더욱 적합한 균주임이 관찰되었다. 즉 재조합 pCold plasmid와 pGro7 plasmid (groES/groEL)로 형질이 전환된 $DH5{\alpha}$가 CalB 발현시스템에 가장 적합하다.

• Journal of Microbiology
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• 제35권2호
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• pp.149-151
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• 1997

A plasmid vector with combined features of yeast shuttle vector and mammalian expression vector was constructed to facilitate expression of cloned gene in both cell-types. All necessary elements required for plasmid maintenance and selection in E. coli, yeast and mammalian cells were size-economically arranged in this plasmid. The numan cytomegalovirus (CMV) immediate early promoter and yeast GAL1 promoter were sequentially placed in front of the gene to be expressed. The synthetic splicing donor and acceptor sequences were inserted into the immediate upstream and downstream of the GAL1 promotor, allowing the CMV promotor to direct the expression of a given gene in mammalian cell environment by splicing out the interfering GAL1 promotor sequence. When the resulting vector containing LacZ as a gene was introduced into yeast and mammalian cells, both cells efficiently produced .betha.-galactosidase, dimonstrating its dual host usage.

• 미생물학회지
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• 제44권2호
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• pp.93-97
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• 2008

최근 Escherichia coli에서 RNA의 분해와 가공과정에 중추적인 역할을 하는 리보핵산 내부분해효소인 RNase E의 효소활성을 조절하는 단백질 조절자인 RraA가 밝혀졌으며, 이 단백질은 E. coli RNase E의 효소활성 부위와 36%의 유사성을 가지는, Streptomyces coelicolor RNase ES의 효소활성을 조절하는 것으로 알려져 있다. S. coelicolor의 유전체에는 RraA와 아미노산 서열이 35.4% 이상 유사한 단백질을 코딩하는 유전자가 두 개 존재하는데, 그 중 하나인 rraAS2를 클로닝하여 E. coli RNase E의 효소활성을 조절하는지를 알아보았다. 그 결과 세포내에서 RraAS2를 발현시키면 RNase E의 과발현에 의해 저해된 세포의 생장을 RraA와 같이 효과적으로는 아니지만, 어느 정도 복원시키는 것을 확인하였다. 또한 RraAS2가 발현됨으로서 RNase E의 과발현에 의해 증가된 ColE1-타입 플라스미드의 복제 수를 14% 감소시키는 것을 관찰하였다. 이러한 결과는 RraAS2가 RNase E의 RNA I분자에 대한 효소 활성을 저해하는 능력을 가지고 있음을 시사한다. 동일한 배양조건에서 E. coli 세포내에서의 RNase E에 대한 RraAS2의 상대적인 발현양이 RraA에 비해 6.2배 낮은 것을 확인하였고, 이로 인해 RraAS2가 RNase E의 과발현에 의한 세포 생장의 저해를 복원하는데 필요한 모든 RNA의 가공과 분해속도를 효과적으로 조절하지는 못한다는 것을 추론할 수 있다.

• Journal of Microbiology and Biotechnology
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• 제18권8호
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• pp.1357-1363
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• 2008

The $P_{entC}$ promoter of the entCERA operon encoding enzymes for enterobactin biosynthesis in Escherichia coli is tightly regulated by the availability of iron in the culture medium. In iron-rich conditions, the $P_{entC}$ promoter activity is strongly repressed by the global transcription regulator Fur (ferric uptake regulator), which complexes with ferrous ions and binds to the Fur box 19-bp inverted repeat. In this study, we have constructed the expression vector pOS2 containing the $P_{entC}$ promoter and characterized its repression, induction, and modulation by quantifying the expression of the lacZ reporter gene encoding $\beta$-galactosidase. $\beta$-Galactosidase activities of E. coli transformants harboring pOS2-lacZ were highly induced in the presence of divalent metal ion chelators such as 2,2'-dipyridyl and EDTA, and were strongly repressed in the presence of excess iron. It was also shown that the basal level $\beta$-galactosidase expression by the $P_{entC}$ promoter was drastically decreased by incorporating the fur gene into the expression vector. Since the newly developed iron chelator-inducible expression system is efficient and cost-effective, it has wide applications in recombinant protein production.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.1
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• pp.226.2-226.2
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• 2003

The FimH subunit of type 1-fimbriated Escherichia coli has been determined as a major cause of urinary tract infection. To produce a possible vaccine antigen against urinary tract infection, the fimH gene was genetically linked to the Itxa2b gene, which was then cloned into the pMAL -p2E expression vector. The chimaeric construction of pMALfimH/Itxa2b was transformed into Escherichia coli TB1 and its N-terminal amino acid sequence was analyzed. (omitted)