• 산업기술연구
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• 제28권B호
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• pp.59-63
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• 2008

Rapid production of therapeutic proteins such as angiostatin and endostatin angiogenic inhibititors has been highly demanded for cancer treatment. In this regard, recombinant human angiostatin and endostatin were successfully expressed as soluble forms by maltose binding protein (MBP)-mediated fusion expression in Escherichia coli. PCR amplified, angiostatin and endostatin genes from human placenta cDNA library were inserted into an expression vector pMAL-c2e to construct prokaryotic expression vectors, pMAL-c2e/AS and pMAL-c2e/ES, respectively. Recombinant angiostatin and endostatin were efficiently expressed in E. coli origami (DE3) after IPTG induction and protein expression were confirmed by SDS-PAGE analyses. The expressed recombinant proteins were purified near homogenity using an amylose affinty column chromatography. In contrast that previous E. coli expressions were all insoluble, our results first time demonstrated that MBP fused human angiostatin and endostatin were soluble in E. coli.

• Genomics & Informatics
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• 제15권4호
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• pp.142-146
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• 2017

More effective production of human insulin is important, because insulin is the main medication that is used to treat multiple types of diabetes and because many people are suffering from diabetes. The current system of insulin production is based on recombinant DNA technology, and the expression vector is composed of a preproinsulin sequence that is a fused form of an artificial leader peptide and the native proinsulin. It has been reported that the sequence of the leader peptide affects the production of insulin. To analyze how the leader peptide affects the maturation of insulin structurally, we adapted several in silico simulations using 13 artificial proinsulin sequences. Three-dimensional structures of models were predicted and compared. Although their sequences had few differences, the predicted structures were somewhat different. The structures were refined by molecular dynamics simulation, and the energy of each model was estimated. Then, protein-protein docking between the models and trypsin was carried out to compare how efficiently the protease could access the cleavage sites of the proinsulin models. The results showed some concordance with experimental results that have been reported; so, we expect our analysis will be used to predict the optimized sequence of artificial proinsulin for more effective production.

• Journal of Microbiology and Biotechnology
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• 제24권2호
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• pp.188-196
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• 2014

Extracellular production of recombinant human bone morphogenetic protein-7 (rhBMP-7) was carried out through the fermentation of Bacillus subtilis. Three significant fermentation conditions and medium components were selected and optimized to enhance the rhBMP-7 production by using the response surface methodology (RSM). The optimum values of the three variables for the maximum extracellular production of rhBMP-7 were found to be 2.93 g/l starch, 5.18 g/l lactose, and a fermentation time of 34.57 h. The statistical optimization model was validated with a few fermentations of B. subtilis in shake flasks under optimized and unoptimized conditions. A 3-L jar fermenter using the shake-flask optimized conditions resulted in a higher production (413 pg/ml of culture medium) of rhBMP-7 than in a shake flask (289.1 pg/ml), which could be attributed to the pH being controlled at 6.0 and constant agitation of 400 rpm with aeration of 1 vvm.

• KSBB Journal
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• 제11권4호
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• pp.438-444
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• 1996

모델 재조합 단백질인 $\beta$galactosidase를 발현하 는 Vaccinia virus를 생산하기 위하여 숙주 동물세 포의 배양조건을 관찰한 결과 감염비 5일 경우 H HeLa는 감염후 60시간 후, HeLa 83는 감염후 40 시간 후에 회수할 때 최 대 의 ${\beta}$-galactosidase 수율 을 얻을 수 있으며, 세포가 대수증식기 일 때 감염하 고 배양온도는 $37^{\circ}C$ 로 하는 것이 최적 배양조건으로 나타났다. 감염후 혈청의 농도는 단백질 수율에 크 게 영향을 미치지는 않으나 3~5% 에서 가장 높은 단백질 수율을 보였으며, 낮은 이온 농도의 용액으 로 세포층을 세척하는 것과 virus 감염시 온도를 20~∼$30^{\circ}C$ 로 낮추는 것은 Vaccinia-HeLa system에서 감염능 증대 효과를 나타내 였다. Dexamethasone 전처리는 HeLa 83에서 ViruS 복제 증대를 HeLa에 서는 virus 복제 감소를 가져왔다.

• Journal of Microbiology and Biotechnology
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• 제14권4호
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• pp.783-788
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• 2004

Marker proteins of genetically-modified organisms (GMO) and their antibodies were prepared and characterized as major components of an analytical system. We selected two GMO markers, neomycin phosphotransferase II and 5- enolpyruvylshikimate-3-phosphate synthase, and produced them from E. coli employing genetic recombination technology. After purification, their structural conformation and binding affinities to the respective antibodies were characterized. The results showed that the recombinant proteins were identical with commercially obtained reference proteins. We further used them as immunogens to raise polyclonal antibodies capable of discriminating GMO containing protein from non-GMO. Well-characterized marker proteins and antibodies will be valuable as immunoreagents in constructing analytical systems such as biosensors and biochips to measure quantities of GMO.

• Molecules and Cells
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• 제25권3호
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• pp.446-451
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• 2008

We assessed heterologous protein expression in 64 strains obtained from the Escherichia coli Reference (ECOR) collection, a collection representing diverse natural E. coli populations. A plasmid generating a glutathione S-transferase and plant carbonic anhydrase fusion protein (GST-CA) under the control of the tac promoter was introduced into the ECOR strains, and the quantity of the fusion protein was determined by SDS-PAGE. The foreign protein was generated at various levels, from very high (40 strains, high producers) to very low (six strains, low producers). Immunoblotting showed that the high producers expressed approximately 250-500 times more GST-CA protein than the low producers. The results of semi-quantitative RT-PCR showed that the low producers generated mRNA levels comparable to those of the high producers, thereby suggesting that, at least in this case, inefficient translation is a major cause of the low production. We introduced a different plasmid, which expressed a maltose binding protein and plant guanylate kinase fusion protein (MBP-GK) into the six low producers. Interestingly, five of these expressed MBP-GK at very high levels. Thus, we conclude that the production of a particular protein from an expression vector can vary considerably, depending on the host strain. Strains in the ECOR collection could function as useful alternative hosts when a desired level of protein expression is not obtained from commonly used strains, such as E. coli K12 or B derivatives.

• KSBB Journal
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• 제15권2호
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• pp.181-187
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• 2000

본 연구에서 갈락토즈를 거의 사용하지 않고 glucose repression 정도가 줄어든 변이주를 이용하여 fed-batch를 통한 발현최적화를 수행하였다. 두 균주에서의 GAL promoter에 의한 외래단백질 생산시 glucose repression 정도에 대해 조사하였는데 대조구 Y2805는 글루코즈가 다 소비된 후 2∼3시간 지난 후 발현이 시작되나 변이주 Y334는 약 0.5 g/L 글루코즈 농도에서 25%정도의 발현이 이루어짐에 따라 변이주 Y334는 GAL promoter에 미치는 glucose repression 정도가 매우 약한 장점을 확인하였다. 배양 중 재조합 미생물인 두균주 변이주 Y334와 대조구 효모 Y2805의 plasmid stability에 대해 안정한 균주임을 알 수 있었으며 대조구 Y2805의 경우도 plasmid stability는 90%로 유지됨을 알 수 있었다. GAL promoter에 의한 외래 단백질 생산시 글루코즈와 갈락토즈, 에탄올의 소비속도를 조사하였는데, 글루코즈와 에탄올의 소비속도는 거의 비슷하였으나 갈락토즈 소비속도는 Y2805는 0.1232 g/L/hr/O.D.이고 변이주 Y334는 0.0131 g/L/hr/O.D. 이다. 또한 mutant Y334와 대조구 효모 Y2805의 Fed-batch 시의 올바른 feeding 방법을 구하기위한 실험을 수행하여 각 균주의 Fed-batch 실험에서의 최적 발현 방법을 획득하였다.

• Journal of Microbiology and Biotechnology
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• 제10권1호
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• pp.115-118
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• 2000

Recombinant plasmids harboring a heterologous gene coding for the enhanced green fluorescent protein (EGFP) were transfected and expressed in Drosophila melanogaster S2 cells. A stable transformation of polyclonal cell populations expressing EGFP were isolated after 4 weeks of selection with hygromycin B. The recombinant EFGP expressed in transformed S2 cells consisted of a molecular weight of 27 kDa. EGFP expression was also confirmed by fluorometric measurement. The maximum EGFP concentration was about 9.3 mg/I. The present findings demonstrate not only the successful stable expression of EGFP in Drosophuila was about 9.3 mgI. The present findings demonstrate not only the successful stable expression of EGFP in Drosophila S2 cells, but also the use of EGFP as a reporter to analyze gene expression, with its potential of a Drosophila cell expression system for recombinant protein production being an alternative to a baculovirus-insect cell expression system.

• Bulletin of the Korean Chemical Society
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• 제32권12호
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• pp.4337-4340
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• 2011

The vitamin D receptor binding peptide, VDRBP, was overexpressed as a fused form with the ubiquitin molecule in Rosetta(DE3)pLysS, a protein production strain of Escherichia coli harboring an induction controller plasmid. The fusion protein was bound to the immobilized metal ions, and the denaturation and renaturation of the fusion protein were performed as a part of the purification procedure. After the elution of the fusion protein, the peptide hormone was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The purity of the resulting peptide fragment was checked by MALDI-TOF mass and NMR spectroscopy. The final yields of the target peptide were around 5 and 2 mg per liter of LB and minimal media, respectively. The recombinant expression and purification of this peptide will enable structural and functional studies using multidimensional NMR spectroscopy and X-ray crystallography.

• 한국어병학회지
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• 제22권3호
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• pp.219-228
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• 2009

The genomic and subgenomic RNAs of fish nodavirus encode the four proteins, protein A, capsid protein, non-structural protein B1 and B2. In this study, we describe the immune response of olive flounder Paralichthys olivaceus immunized with live fish nodavirus or recombinant capsid protein, non-structural protein B1 and B2 expressed in E. coli. Nodavirus-infected flounder produced antibodies to capsid protein, B1 and B2 and nodavirus-neutralizing activities were detected in the serum of the nodavirus-infected flounder. The flounder were immunized against the three recombinant proteins of fish nodavirus and the sera from these immunized fishes were assayed for nodavirus-specific antibody by ELISA and a neutralization test. In the immunized flounder, all three recombinant proteins induced the production of similar levels of antibody, but only the antibody to capsid protein significantly neutralized nodavirus. These results indicate that all three nodaviral proteins are immunogenic in flounder, but only the capsid protein can induce neutralizing antibody against nodavirus.