• 공업화학
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• 제17권5호
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• pp.557-560
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• 2006

Aspergillus niger LK의 epoxide hydrolase (EH)를 codon usage를 고려한 Escherichia coli 균주에서 고효율로 발현할 수 있었다. E. coli에서 잘 사용되지 않는 rare codon에 대한 tRNA 유전자 정보가 들어있는 plasmid를 함유한 E. coli 균주인 Rosetta (DE3)PLysS를 숙주세포로 사용하였다. A. niger EH를 발현시킨 재조합 E. coli를 생촉매로 사용하여 라세믹 styrene oxide 혼합물과 반응시켰을 때, (R)-styrene oxide에 대한 입체선택적 가수분해활성이 향상됨을 확인할 수 있었다. 또한 라세믹 기질로부터 입체적으로 고순도인 99% ee 값을 갖는 광학적으로 순수한 (S)-styrene oxide를 얻을 수 있었다.

• 생명과학회지
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• 제16권3호
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• pp.415-419
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• 2006

방향족 에폭사이드 기질에 대한 입체선택적 가수분해능이 우수한 Rhodotorula glutinis의 epoxide hydrolase (EH)를 codon usage를 고려한 Escherichia coli 균주에서 고효율로 발현할 수 있었다. 효모인 R. glutinis와 박테리아인 E. coli에서의 codon usage 선호도를 분석하고 그 차이를 고려하여 E. coli 에서 잘 사용되지 않는 rare codon에 대한 tRNA유전자정보가 들어 있는 pRARE plasmid를 함유한 E. coli 균주인 Rosetta(DE3)pLysS를 숙주세포로 사용하였다. R. glutinis EH를 발현시킨 재조합 E. coli를 생촉매로 사용하여 라세믹 styrene oxide 혼합물과 반응시켰을 때, (R)-styrene oxide에 대한 입체선택적 가수분해활성이 wild type R. glutinis 대비 매우 향상됨을 관찰할 수 있었다. 또한 라세믹 기질로부터 입체적으로 고순도인 99% ee 값을 갖는 광학적으로 순수한 (S)-styrene oxide를 얻을 수 있었다.

• Animal cells and systems
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• 제16권6호
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• pp.455-461
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• 2012

Amongst the various antimicrobial peptides, lysozyme plays a central role in initiating and maintaining the antibacterial defense response of insect. Here we propose the biosynthesis and refolding of recombinant lysozyme in Escherichia coli expressed in inclusion body form. The Agrius lysozyme gene was amplified using gene specific primers and then ligated into the pGEX-4T-1 vector, which contained the glutathione S-transferase (GST) gene as a fusion partner. A recombinant lysozyme was expressed in E. coli Rosetta cells using a pGEX-4T-1 expression vector, and the fusion protein was induced by ioporpyl-${\beta}$-D-thiogalactopyranoside (IPTG). The recombinant protein produced as an inclusion body was resolubilized in solubilization buffer, and the resultant solution was dialyzed in refolding buffer. After thrombin cleavage, the recombinant lysozyme was purified by ion exchange chromatography and reverse phase chromatography. The recombinant lysozyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoreactivity against the anti-Agrius lysozyme was observed by western blot analysis of this protein. The recombinant lysozyme displayed antibacterial activity against Bacillus megaterium and Micrococcus luteus, which was confirmed by the inhibition zone assay.

• 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.

• Asian Pacific Journal of Cancer Prevention
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• 제15권12호
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• pp.5055-5061
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• 2014

In this study, an anti-oxidant and anti-tumor protein Latcripin-3 of Lentinula edodes C91-3 was expressed in Escherichia coli. for the first time. According to the cDNA library, the full-length gene of Latcripin-3 was cloned by the methods of 3'-full rapid amplification of cDNA Ends (RACE) and 5'-full RACE. The structural domain gene of Latcripin-3 was inserted into the pET32 a(+). The functional protein of Latcripin-3 was expressed in Rosetta-gami (DE3) E. coli, evaluated by Western blotting and mass spectrometry. DPPH testing showed that the protein Latcripin-3 can scavenge free radicals remarkably well. The activity of functional protein Latcripin-3 on A549 cells was studied with flow cytometry and the MTT method. The MTT assay results showed that there was a decreases in cell viability in a dose-dependent and time-dependent manner in protein Latcripin-3 treated groups. Flow cytometry demonstrated that Latcripin-3 can induce apoptosis and block S phase dramatically in human A549 lung cancer cells as compared to the control group. At the same time, the cell ultrastructure observed by transmission electron microscopy supported the results of flow cytometry. This research offers new insights and advantages for identifying anti-oxidant and anti-tumor proteins.

• Journal of Microbiology and Biotechnology
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• 제26권11호
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• pp.1881-1890
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• 2016

Manganese superoxide dismutase (MnSOD) is a vital enzyme that protects cells from free radicals through eliminating superoxide radicals ($O^{2-}$). Hirudin, a kind of small active peptide molecule, is one of the strongest anticoagulants that can effectively cure thrombus diseases. In this study, we fused Hirudin to the C terminus of human MnSOD with the GGGGS linker to generate a novel dual-feature fusion protein, denoted as hMnSOD-Hirudin. The hMnSOD-Hirudin gene fragment was cloned into the pET15b (SmaI, CIAP) vector, forming a recombinant pET15b-hMnSOD-Hirudin plasmid, and then was transferred into Escherichia coli strain Rosetta-gami for expression. SDS-PAGE was used to detect the fusion protein, which was expected to be about 30 kDa upon IPTG induction. Furthermore, the hMnSOD-Hirudin protein was heavily detected as a soluble form in the supernatant. The purification rate observed after Ni NTA affinity chromatography was above 95%. The hMnSOD-Hirudin protein yield reached 67.25 mg per liter of bacterial culture. The identity of the purified protein was confirmed by western blotting. The hMnSOD-Hirudin protein activity assay evinced that the antioxidation activity of the hMnSOD-Hirudin protein obtained was $2,444.0{\pm}96.0U/mg$, and the anticoagulant activity of the hMnSOD-Hirudin protein was $599.0{\pm}35.0ATU/mg$. In addition, in vitro bioactivity assay showed that the hMnSOD-Hirudin protein had no or little cytotoxicity in H9c2, HK-2, and H9 (human $CD_4{^+}$, T cell) cell lines. Transwell migration assay and invasion assay showed that the hMnSOD-Hirudin protein could suppress human lung cancer 95-D cell metastasis and invasion in vitro.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• 제13권2호
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• pp.134-145
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• 2010

목 적: 로타바이러스 감염역학의 변화를 연구하기 위해 A군 로타바이러스의 VP6 유전자를 대장균에 발현시켜 확보한 rVP6 단백질이 항원성이 있는지를 확인하고 이것을 항원으로 한 효소면역측정법이 로타바이러스 IgG, IgA와 IgM 항체를 정량적으로 평가할 수 있는지 확인하고자 하였다. 방 법: 경상대학교병원에서 로타바이러스 감염을 진단받은 소아들 중 진단 받기 전, 진단 당시, 회복기 이후의 연속적인 혈청을 확보할 수 있었던 22명에게서 100개의 혈청을 경상대학교병원 인체자원은행으로부터 제공받아 로타바이러스 VP6 유전자를 클로닝 하여 대장균에 발현시켜 제조한 rVP6 항원으로 한 효소면역측정법으로 IgG, IgA와 IgM 항체 역가를 측정하였다. 이 중 건강한 신생아 4명에서는 면역 블로팅을 같이 시행하였다. 결 과: 건강한 신생아와 영유아 17명에서 감염 후 확보된 혈청에서 IgG, IgA, IgM 항체 중 최소한 한 종류의 항체 역가 증가가 동반되어 있었다. 면역이 저하된 소아 5명 중 4명에서는 IgG 항체 역가는 증가되었으나 IgA 항체 역가는 2명에서만 증가하였고, IgM 항체 역가는 5명 모두 증가하지 않았다. 신생아 4명에서 시행된 면역 블로팅 검사에서는 IgM 항체인 경우는 효소면역측정법보다 예민하게 진단 초기부터 4명 모두 양성으로 판정되었다. 결 론: A군 로타바이러스의 VP6 유전자를 대장균에 발현시켜 확보한 rVP6 단백질은 항원성이 있으며 이것을 항원으로 한 효소면역측정법은 로타바이러스 감염후 IgG, IgA, IgM 항체 역가 증가를 정량적으로 평가할 수 있어 지역사회에서 발생한 로타바이러스 감염역학의 변화를 연구하는데 유용할 것으로 판단된다.

• Parasites, Hosts and Diseases
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• 제52권1호
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• pp.93-97
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• 2014

Subolesin (4D8), the ortholog of insect akirins, is a highly conserved protective antigen and thus has the potential for development of a broad-spectrum vaccine against ticks and mosquitoes. To date, no protective antigens have been characterized nor tested as candidate vaccines against Dermacentor silvarum bites and transmission of associated pathogens. In this study, we cloned the open reading frame (ORF) of D. silvarum 4D8 cDNA (Ds4D8), which consisted of 498 bp encoding 165 amino acid residues. The results of sequence alignments and phylogenetic analysis demonstrated that D. silvarum 4D8 (Ds4D8) is highly conserved showing more than 81% identity of amino acid sequences with those of other hard ticks. Additionally, Ds4D8 containing restriction sites was ligated into the pET-32(a+) expression vector and the recombinant plasmid was transformed into Escherichia coli rosetta. The recombinant Ds4D8 (rDs4D8) was induced by isopropyl ${\beta}$-D-thiogalactopyranoside (IPTG) and purified using Ni affinity chromatography. The SDS-PAGE results showed that the molecular weight of rDs4D8 was 40 kDa, which was consistent with the expected molecular mass considering 22 kDa histidine-tagged thioredoxin (TRX) protein from the expression vector. Western blot results showed that rabbit anti-D. silvarum serum recognized the expressed rDs4D8, suggesting an immune response against rDs4D8. These results provided the basis for developing a candidate vaccine against D. silvarum ticks and transmission of associated pathogens.