• 한국미생물·생명공학회지
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• 제25권2호
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• pp.137-143
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• 1997

Porcine transforming growth factor-$\beta$1 (TGF-$\beta$1) was expressed in Escherichia coli using cDNA of TGF-$\beta$1 and glutathione S-transferase (GST) fusion vector pGEX-1$\lambda$T. An ApoI-Tth111I fragment of cDNA which correspond to the amino acid residues from 123 to 390 of the precursor TGF-$\beta$1 was inserted into EcoRI-Tth111I digested pGEM#-l$\lambda$T and the recombined plasmid was named pGET-12. Gene products from the cloned regions of the recombinant plasmids pGET-12 was not detected in soluble fraction of cell free extract but detected in insoluble fraction. The solubilization of insoluble gene product was achieved by the treatment of N-laurylsarcosine. Molecular weight of partially purified proteins determined by electrophoresis was same as expected from cloned fragment. The ELISA test results of the purified proteins showed that immunologically detectable epitope was preserved in recombinant protein.

• Journal of Microbiology
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• 제35권2호
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• pp.123-126
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• 1997

The aim of the present study was to optimize culture condition for the expression of lipocortin-1$_{1-185}$ in a recombinant of Escherichia coli using batch system. Plasmid (pHT22) carrying lipocortin-1$_{1-185}$ gene was well maintained in the recombinant with the addition of amplicillin as a selection pressures. Optimum temperature was 28$^{\circ}C$ for seed culture and 4$0^{\circ}C$ for main culture and the optimum pH was 7.0. The production of Lipocortin-1$_{1-185}$ was closely associated with cell growth and related to plasmid amplification.

• 생명과학회지
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• 제20권6호
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• pp.934-939
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• 2010

박테리오신의 항균활성 개선을 위해 선행연구에서 개발된 ADH 프로모터에 의해 박테리오신 유전자를 발현시키는 효모발현 벡터에 GPD 프로모터 단편을 도입하여 강력한 프로모터를 가진 효모발현 재조합 플라스미드DNA를 제작하였다. ADH 프로모터에 의해 박테리오신 유전자를 발현시키는 기존의 형질전환 효모들에 비해 새롭게 개발된 GPD 프로모터에 의해 박테리오신 유전자를 발현시키는 형질전환 효모들이 그람양성 대표세균인 고초균(B. subtilis)과 그람음성 장내세균인 대장균(E. coli) 모두에서 보다 높은 생육억제환을 나타내는 것을 확인 하였다. 이 연구의 결과로 GPD 프로모터에 의해 발현이 유도되는 박테리오신 생산 효모들을 이용하여 부패하기 쉬운 식품들의 보존성을 향상시키기 위한 보존제 대체물질 또는 가축 사료에서 병원균의 생육을 저해하기 위한 항생제 대체물질의 산업적인 생산이 가능하게 될 것으로 기대된다.

• 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.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 1991년도 춘계학술발표대회 논문집
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• pp.237-242
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• 1991

In order to increase the production of glutathione by maximizing the expression of recombinant gsh plasmids, two genes responsible for the biosynthesis of glutathione were cloned. A gshI gene was cloned onto pBR322 plasmid as 3.6Kb PstI DNA fragment from E. coli K-12 chromosomal DNA. Also gshII gene was cloned onto pUC13 plasmid as 2.2Kb PstI-BamHI DNA fragment. In order to improve the glutathione producing activity more efficiently, various recombinant plasmids containing tandem repeated gshI genes or both genes in various copy number onto the same vector were constructed. E. coli cells harboring pGH501 plasmid (pUC8-gshI$\cdot$I$\cdot$II) showed the highest glutathione synthesizing activity. The conditions for glutathione production with an ATP-generating system such as acetate kinase reaction of E. coli cells or glycolytic pathway of yeast cells were examined using the E. coli cells harboring the pGH501 plasmid. When the acetate kinase reaction of E. coli cells was used as an ATP generating system, 20mM of L-csteine was converted into glutathione with a yield of $100\%$.

• 한국미생물·생명공학회지
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• 제17권3호
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• pp.188-192
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• 1989

토양에서 분리된 알카리 내성 Bacillus sp. YA-14의 chromosomal DNA로부터 B. subtilis 207-25 에 cloning된 promoter 유전자들을 그 공여 균주로 재형질전환시킬 수 있었다. 그 결과 promoter 유전자들은 공여 균주내에서 대부분의 특성들이 B. subtilis 207-25 내에서와 유사하였다. 재조합 plasmid p-12, p-l2Bl 및 p-l2B2의 CAT 비활성은 B. subtilis 207-25의 형질전환체보다 약간 높게 나타났으며 반면 pPL708의 CAT 비활성은 Bacillus sp. YA-14에서 오히려 상대적으로. 낮게 나타났다. Chloramphenicol에 의한 induction 정도도 월등하게 높은 것으로 나타나 이를 이용한 expression vector의 유용성이 높은 것으로 생각되었다.

• 대한수의학회지
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• 제39권4호
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• pp.786-796
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• 1999

As an attempt to develop an effective control method against theileriosis, recombinant antigen protein was produced. Thirty-two kDa membrane protein(MP) gene of T sergenti was amplified through RT-PCR from extracted total RNA of T sergenti isolated in Chonbuk, Korea. The amplified 869 bp of Korean T sergenti membrane gene was cloned and the base sequences were analyzed. The amplified gene was cloned into E coli expression vector, pQE32 plasmid vector, and the vector was introduced into E coli strain M15 to produce the recombinant membrane protein. For the induction of T sergenti membrane protein(KTs-MP), the plasmid harboring E coli strain M15 were cultured in the presence of IPTG, and the recombinant protein were purified by $Ni^+$-NTA agarose. Then, to confirm the authenticity of the produced membrane protein, molecular weight of expressed recombinant KTs-MP was analyzed by SDS-PAGE and Western blotting. The molecular weight of expressed recombinant protein was 32 kDa as expected. The recombinant KTs-MP was successfully recognized by anti-His Tag antibody, antisera of T sergenti infected cattle and monoclonal antibody of T sergenti membrane protein. Therefore, we concluded that the authentic 32 kDa membrane protein of T sergenti was produced as immunologically recognizable form.

• Journal of Periodontal and Implant Science
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• 제38권sup2호
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• pp.343-354
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• 2008

Purpose: Aggregatibacter actinomycetemcomitans is associated with localized aggressive periodontitis. It produces cytolethal distending toxin (CDT), which induces cell cycle arrest in the G2/M phase. The CDT holotoxin is composed of CdtA, CdtB, and CdtC. CdtB has structural homology to human DNase I and is an active component of the CDT complex acting as a DNase. In particular, the pattern homology seen in the CdtB subunit has been associated with specific DNase I residues involved in enzyme catalysis, DNA binding, and metal ion binding. So, to study the functions and regulation of recombinant CdtB, we made up a quantity of functional recombinant CdtB and tested it in relation to the metal ion effect. Materials and Methods: We constructed the pET28a-cdtB plasmid from A. actinomycetemcomitans Y4 by genomic DNA PCR and expressed it in the BL21 (DE3) Escherichia coli system. We obtained the functional recombinant CdtB by the refolding system using the dialysis method and then analyzed the DNase activity and investigated the metal ion effect from plasmid digestion. Results: The recombinant CdtB subunit was expressed as the inclusion bodies. We were able to obtain functional recombinant CdtB subunit using refolding system. We confirmed that our refolded recombinant CdtB had DNase activity and was influenced by the metal ions $Mg^{2+}$ and $Ca^{2+}$. Conclusion: We suggest that the factors influencing recombinant CdtB may contribute to CDT associated diseases, such as periodontitis, endocarditic, meningitis, and osteomyelitis.

• Journal of Animal Science and Technology
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• 제50권2호
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• pp.177-184
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• 2008

Acid-labile subunit(ALS)는 85-kDa 크기의 당단백질로서 7.5-kDa의 insulin-like growth factor(IGF) 및 40~45-kDa IGF-binding protein-3와 결합하여 150-kDa ternary complex를 형성하는 혈장단백질이다. 선행연구에서 본 연구진은 reverse transcription-polymerase chain reaction(RT-PCR) 방법으로 돼지(porcine) ALS(pALS)의 coding sequence를 합성하여 plasmid vector에 삽입시켜 ‘expression construct’를 제작한 바 있다. 그러나 본 expression construct의 pALS coding sequence에는 PCR error로 추정되는 원인으로 말미암아 2개의 bases에서 mis-sense mutation이 일어난 것이 발견되었다. 본 연구에서는 ‘site-directed mutagenesis’ 방법으로 pALS의 올바른 coding sequence를 합성하여 ‘insert DNA’의 마지막 codon 다음에 ‘His-tag’ sequence가 위치한 pET- 28a(+) plasmid expression vector에 삽입하였다. 본 expression construct는 E. coli BL21(DE3) 세포에서 ‘induction’ 시켰고, 발현된 유전자재조합(recombinant) peptide는 Ni-affinity chromato- graphy로 정제하였다. 이렇게 affinity chro- matography로 정제된 peptide는 SDS-PAGE에서 66kDa 위치에 single band를 나타냄으로써 recombinant pALS의 예상된 질량과 일치하였다. 이상의 결과는 본 연구에서 recombinant pALS peptide가 성공적으로 발현정제되었음을 시사한다.

• 생명과학회지
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• 제11권4호
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• pp.385-391
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• 2001

Filamentous hemagglutinin (FHA) is considered as an essential immunogenic component for incorporation into acellular vaccines against Bordetella pertussis, the causative agent of whooping cough. Classically, antipertussis vaccination has employed an intramuscular route. An alternative approach to stimulate mucosal and systemic immune responses is oral immunization with recombinant live vaccine carrier strains of Salmonella typhimurium. An attenuated live Salmonella vaccine sgrain($\Delta$cya $\Delta$crp) expressing recombinant FHA(rFHA) was developed. Stable expressionof rFHA was achieved by the use of balanced-lethal vector-host system. which employs an asd deletion in the host chromosome to impose in obligate requirement for diaminopimelic acid. The chromosomal $\Delta$asd mutation was complemented by a plasmid vector possessing the asd$^{+}$ gene. A 3 kb DNA fragment encoding immuno dominant regionof FHA was subcloned in-frame downstream to the ATG translation initiation codon in the multicopy Asd$^{+}$ pYA3341 vector to create pYA3457. Salmonella vaccine harboring pYA3457 expressed approximately 105kDa rFHA protein. The 100% maintenance of [YA3457 in vaccine strain was confirmed by stability examinations. Additionally, a recombinant plasmid pYA3458 was constructed to overpress His(8X)-tagged rFHA in Essherichia coli. His-tagged rFHA was purified from the E. coli strain harboring pYA3458 using Ni$^{2+}$-NTA affinity purification system.>$^{2+}$-NTA affinity purification system.