• 공업화학
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• 제23권6호
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• pp.599-603
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• 2012

위치특이적 돌연변이 기술을 이용하여 활성을 향상시킨 Mugil cephalus 유래 에폭사이드 가수분해효소(epoxide hydrolase, McEH)를 발현하는 재조합 대장균 균주를 생촉매로 이용하여 유기용매상에서 라세믹 스틸렌 옥사이드(styrene oxide)에 대한 입체선택적 가수분해 반응을 수행하였다. 재조합 균주의 세포 성장 시간대별로 McEH 단백질의 발현유도 특성을 분석하였으며, 대수성장기에 IPTG를 첨가했을 때 생촉매 활성이 2.2배 향상되었다. 유기용매의 Log P 값이 증가할수록 생촉매의 입체선택적 가수분해 활성이 증가하였으며, 이소옥탄(isooctane)을 반응용매로 사용하였을 때 생촉매의 입체선택적 가수분해 활성이 가장 우수하였다. 동결건조과정에서 skim milk 또는 sucrose와 같은 lyoprotectant를 사용하여 활성 안정화를 시킨 재조합 대장균 생촉매를 사용하여 이소옥탄에서 20 mM 라세믹 스틸렌 옥사이드에 대하여 광학순도 98%의 (S)-스틸렌 옥사이드를 이론수율 대비 53.6%로 제조할 수 있었다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제1권1호
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• pp.9-12
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• 1996

In order to produce the single chain precursor of a novel human insulin analogue, (B30-Homoserine) insulin, the fermentative behaviors of Escherichia coli JM103 were studied, which harbors pKBA plasmid carrying a hybrid gene in which the gene for a single chain precursor was fused with lacZ gene under tac promoter. The maximal induction of gene expression was achieved when more than 0.05 mM of isopropyl-$\beta$-D-thiogalactopyranoside(IPTG) was supplemented to fermentation medium after 4 h cultivation of E. coli, and followed by longer than 2-h fermentation. The hybrid protein of the single chain insulin precursor was isolated from cytoplasmic inclusion bodies by dissolving in 8M urea solution, and purified through DEAE-Sephacel and Sephadex G-200 column chromatographies with a recovery of 35%. The finally purified hybrid protein showed a single band on sodium dodecyl sulfate-polyacrylamide gel.

• International Journal of Industrial Entomology and Biomaterials
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• 제16권2호
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• pp.87-92
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• 2008

Bombyx mori nucleopolyhedrovirus (BmNPV) orf47 gene was characterized for the first time. The coding sequence of Bm47 was amplified and subcloned into the prokaryotic expression vector pET-30a(+) in order to produce His-tagged fusion protein in the BL21 (DE3) cells. The His-Bm47 fusion protein was expressed efficiently after induction with IPTG. The purified fusion protein was used to immunize New Zealand white rabbits to prepare polyclonal antibody. As the genome of BmNPV is available in GenBank and the EST database of BmNPV is expanding, identification of novel genes of BmNPV was conceivable by data-mining techniques and bioinformatics tools. Structural bioinformatics approach to analyze the properties of Bm47 encodes protein.

• 한국미생물·생명공학회지
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• 제28권1호
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• pp.21-25
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• 2000

We have got several clones from Serratia marcescens which stimulated the cells to use maltose as a carbon source in Escherichia. coli TP2139 ( lac, crp). One of the cloned genes, pCKB17, was further analyzed. In order to find whether the increased expression of the gent was under the direction of maltose metabolism, we constructed several recombinant subclones. We have found that the clone, pCKB17AV, codes maltose metabolism stimulation(mms) gene. E. coli transformed with the cloned gene showed increase in the activity of maltose utilzation, The recombinant proteins expressed by multicopy and induction with IPTG, one polypeptide of 29-kDa, was confirmed by SDS-PAGE. The overexpression of maltose-binding proter protein in the presence of mms gene was confirmed by Western blot analysis. Southern hybridization analysis confirmed that the cloned DNA fragment was originated from S. marcescens chromosomal DNA.

• Journal of Microbiology and Biotechnology
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• 제8권1호
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• pp.92-95
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• 1998

Overproduction of Escherichia coli thiol peroxidase in the periplasmic space was achieved by locating the appropriate gene on a downstream region of the strong T7 promoter. E. coli strain BL21 carrying the recombinant plasmid pSK-TPX was induced by IPTG, lysed, and analyzed by SDS-polyacrylamide gel electrophoresis. A large amount of the overexpressed thiol peroxidase was located in the periplasmic space. A homogeneous thiol peroxidase was obtained from E. coli osmotic shock fluid by simple one-step gel permeation chromatography.

• 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.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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• pp.183-186
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• 2000

Recombinant E. coli JM109(pZH3-5/pMT) harboring manganese transport gene(mntA) and metal sequestering protein, metallothionein(MT), was cultivated to accumulate cadmium in aqueous phase. Bioaccumulation followed Michaelis-Menten type kinetics. Equilibrium isotherm showed Langmuir type isotherm. IPTG induction cell showed fast $Cd^{2+}$ uptake and had higher uptake rate than wild type and no-induced cell. The optimum pH and temperature for $Cd^{2+}$ uptake was 7 and $37^{\circ}C$, respectively. Manganese (0.01M) inhibited the $Cd^{2+}$ accumulation. However, $Cu^{2+}$, $Zn^{2+}$ and $Pb^{2+}$ did not affect the $Cd^{2+}$ bioaccumulation.

• Bulletin of the Korean Chemical Society
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• 제32권7호
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• pp.2405-2409
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• 2011

The psychrophilic bacteria Psychromonas arctica survives at subzero temperatures by having adapted several protective mechanisms against freezing and oxidative stresses. Many reactive oxygen species are likely generated in P. arctica as a result of reduced metabolic turnover rates. A previous study identified the pasod gene for superoxide dismutase from P. arctica using a series of PCR amplifications. Here, upon cloning into a His-tag fused plasmid, the sod gene from P. arctica (pasod) was successfully expressed by IPTG induction. His-tagged PaSOD was subsequently purified by $Ni^{2+}$-NTA affinity chromatography. The purified PaSOD exhibited a higher SOD activity than that of Escherichia coli (EcSOD) at all temperatures. The difference in activity between PaSOD and EcSOD becomes even more significant at 4$^{\circ}C$, indicating that PaSOD plays a functional role in the cold adaptation of P. arctica in the Arctic.

• Journal of Microbiology and Biotechnology
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• 제18권5호
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• pp.926-932
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• 2008

Human heavy chain (H-) and light chain (L-) ferritins were amplified from a human cDNA library. Each ferritin gene was inserted downstream of the T7 promoter of bacterial expression vectors, and two types of coexpression vectors were constructed. The expression levels of recombinant ferritins ranged about 26-36% of whole-cell protein. H-ferritin exhibited a lower expression ratio compared with L-ferritin, by a coexpression system. However, the coexpression of HL-ferritins was significantly increased above the expression ratio of H-ferritin by cultivation without IPTG induction overnight. Purified recombinant H-, L-, HL-, and LH-ferritins were shown to be homo- and heteropolymeric high molecular complexes and it was indicated that their assembled subunits would be able to work functionally in the cell. Thus, these results indicate an improvement in the expression strategy of H-ferritin for heteropolymeric production and studies of ferritin assembly in Escherichia coli.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
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• pp.160.1-160.1
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• 2003

In order to confirm the biological function of ORF10 in cephabacin biosynthesis gene cluster of Lysobacter lactamgenus as an ATP-binding-cassette (ABC) transporter, the gene for ORF10 was amplified and subcloned into pET-28a(+) expression vector. After gene induction with 0.5 mM IPTG at 30~! and further cultivation at $30^~$ !. for 8 hr, a lot of the recombinant ORF10 protein was produced as soluble form in cytoplasmic fraction as well as a membrane protein in the membrane fraction as likely as other ABC transporters. (omitted)