• 한국미생물·생명공학회지
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• 제23권6호
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• pp.784-788
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• 1995

The lambda Integrase (Int) carries out site-specific recombination between the two partner DNA sequences, attachment P (attP) and attachment B (attB). In order to study the recombination mechanism, a large quantity of pure integrase is required. Then, we constructed an int gene inserted recombinant plasmid (pNYL3) by using the pQE31 HIS-Tag vector, and produced the fusion protein, 6xHIS-Int from the E. coli TG1 strain carrying the pNYL3 plasmid. The recombinant protein produced was purified by phosphocellulose and Ni$^{++}$-NTA affinity column chromatographies. The result of the in vitro recombination assay using the standard reaction mixture containing 6xHIS-Int and partially purified integration host factor (IHF) showed that the 6xHIS-Int tagged recombination Integrase had the full recombination activity.

• 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.

• BMB Reports
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• 제43권5호
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• pp.319-324
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• 2010

Experimental bioinformatics data obtained from an E. coli cell-based eukaryotic protein purification experiment were analyzed in order to identify any bottleneck as well as the factors affecting the target purification. All targets were expressed as His-tagged maltose-binding protein (MBP) fusion constructs and were initially purified by immobilized metal affinity chromatography (IMAC). The targets were subsequently separated from the His-tagged MBP through TEV protease cleavage followed by a second IMAC isolation. Of the 743 total purification trials, 342 yielded more than 3 mg of target proteins for structural studies. The major reason for failure of target purification was poor TEV proteolysis. The overall success rate for target purification decreased linearly as cysteine content or isoelectric point (pI) of the target increased. This pattern of pI versus overall success rate strongly suggests that pI should be incorporated into target scoring criteria with a threshold value.

• International Journal of Industrial Entomology and Biomaterials
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• 제21권2호
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• pp.157-162
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• 2010

Open reading frame 35 (bm35) of the Bombyx mori nucleopolyhedrovirus (BmNPV) is a special gene whose homologues are only found in some group-I nucleopolyhedroviruses, suggesting that bm35 plays a specific role in the viral life cycle. This paper described the characterization of BmNPV bm35. Computerassisted sequence analysis shows that a putative RING finger motif is observed in the protein, Bm35 encoded by bm35. The coding sequence of bm35 was amplified and subcloned into the vector pET30a(+) and the $(His)_6$-tagged fusion protein His-Bm35 was expressed in the Escherichia coli BL21 (DE3) LysS cells. The bm35 transcript and Bm35 protein were detected in BmNPV-infected BmN cells at 12~48 h post infection (p.i.) by RT-PCR and Western blot analysis using the polyclonal antibody generated by immunizing a rabbit with purified $(His)_6$-tagged Bm35, suggesting that bm35 is synthesized in the late stage of BmNPV infection cycle. Bm35 was not a structural component associated with budded virus (BV) and occlusion derived virus (ODV). These data indicated that bm35 is a functional gene in the BmNPV life cycle.

• International Journal of Industrial Entomology and Biomaterials
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• 제22권2호
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• pp.59-64
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• 2011

Protein kinase C (PKC) is involved in many cellular signaling pathways, it participates in many physiological processes, such as cell cycle, growth, proliferation, differentiation and apoptosis. To investigate the effect of PKC on the silkworm midgut tissue infection of Bombyx mori parvo-like virus (BmPLV), a B. mori atypical protein kinase C (BmaPKC) gene was cloned from larval midgut tissue, expressed in E. coli and purified. Additionally, the BmPLV susceptible silkworm strain and resistant silkworm strain were used to test the effect of the B. mori infection on BmPLV. The result showed that BmaPKC encodes a predicted 586 amino acid protein, which contains a C-terminal kinase domain and an N-terminal regulatory domain. The maximum expression amount of the soluble (His)6-tagged fusion protein was detected after 0.8 mmol/L IPTG was added and cultured at $21^{\circ}C$. The (His) 6-tagged fusion protein revealed about 73 kDa molecular weight which confirmed by western blot and mass spectrography. Furthermore BmaPKC protein were detected at 0-72 h post-infection in BmPLVinfected larval midgut tissue, western blot showed that as time went on, the expression of BmaPKC increased gradually in susceptible strain, the expression quantity on 72 h is 5 times of 0 h. However, in resistant strain, the expression quantity is slightly lower than susceptible strain. But no significant change in resistant strain was observed as time went on. The available data suggest that BmaPKC may involve in the regulation of BmPLV proliferation.

• The Plant Pathology Journal
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• 제25권1호
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• pp.47-53
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• 2009

Sequence analysis of the Chlorella virus SS-2 revealed one putative nuclease gene that is 807 bp long and encodes a 31kDa protein. Multiple sequence alignment analysis reveals the presence of highly conserved PD-(D/E)XK residues in the encoded protein. The gene cloned into an expression vector was expressed as a His-tagged fusion protein in chaperone containing pKJE7 cells. The recombinant protein was purified using a His-Trap chelating HP column and used for functional analysis. Exonuclease activity of the SS-2 nuclease was detected when the DNA substrates, such as linear ssDNA, PCR amplicon, linear dsDNA with 5'-overhang ends, 3'-overhang ends, or blunt ends were used. Covalently closed circular DNA was also degraded by the SS-2 recombinant protein, suggesting that the SS-2 nuclease has an endonuclease activity. Stable activity of SS-2 nuclease was observed between $10^{\circ}C$ and $50^{\circ}C$. The optimum pH concentrations for the SS-2 nuclease were pH 6.0-8.5. Divalent ions inhibited the SS-2 nuclease activity.

• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2007년도 제48회 학술심포지움 및 추계국제학술대회
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• pp.111.1-111.1
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• 2007

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• Journal of Microbiology and Biotechnology
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• 제24권7호
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• pp.998-1003
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• 2014

Aspartate aminotransferase (AST; E.C. 2.6.1.1), a vitamin B6-dependent enzyme, preferentially promotes the mutual transformation of aspartate and ${\alpha}$-ketoglutarate to oxaloacetate and glutamate. It plays a key role in amino acid metabolism and has been widely recommended as a biomarker of liver and heart damage. Our study aimed to evaluate the extensive preparation of AST and its application in quality control in clinical laboratories. We describe a scheme to express and purify the 6His-AST fusion protein. An optimized sequence coding AST was synthesized and transformed into Escherichia coli BL21 (DE3) strain for protein expression. Ideally, the fusion protein has a volumetric productivity achieving 900 mg/l cultures. After affinity chromatography, the enzyme activity of purified AST reached 150,000 U/L. Commutability assessment between the engineered AST and standard AST from Roche suggested that the engineered AST was the better candidate for the reference material. Moreover, the AST showed high stability during long-term storage at $-20^{\circ}C$. In conclusion, the highly soluble 6His-tagged AST can become a convenient tool for supplying a much better and cheaper standard or reference material for the clinical laboratory.

• 생명과학회지
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• 제28권1호
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• pp.78-82
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• 2018

본 연구에서는 미생물 검출용 바이오센서 제작을 위해 재조합 람다 파아지 꼬리 단백질 J을 센싱 요소로 활용가능한지를 조사하였다. 융합 단백질의 입체 장애를 최소화하기 위해 J 단백질 절편의 N-말단을 6X His-tag로 융합하고 HisTALONTM 컬럼으로 정제하였다. 정제 단백질은 약 38 kDa 크기를 SDS-PAGE에서 나타내며 anti-His 단클론 항체와 반응하였다. Anti-His 단클론 항체는 6HN-J를 처리한 E. coli K-12와 특이적으로 결합하나 BSA 처리하거나 6HN-J 처리한 다른 미생물들(Salmonella typhimurium, Bacillus subtilis, Pseudomonas aeruginosa)과는 결합되지 않음을 보여주었다. 또한 정제 단백질과 숙주세포의 결합은 온전한 람다 파아지의 in vivo 숙주 표면 흡착을 약 $1{\mu}g/ml$ 단백질 농도에서 50%, $25{\mu}g/ml$ 단백질 농도에서 거의 완전히 방해하였다. 결론적으로 재조합 6HN-J 단백질은 탁월한 선택성과 선별성으로 인해 바이오센서의 제작에서 센싱 요소로 활용 가능함을 시사한다.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2001년도 Proceedings of 2001 International Symposium
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• pp.135-136
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• 2001

The Tk-ptp gene encoding a protein tyrosine phosphatase (PTPase) from the hyperthermophilic archaeon Thermococcus kodakaraensis KODI was cloned and sequenced. Sequence analysis indicated that Tk-ptp encoded a protein consisting 147 amino acid residues (16,953 Da). The wild type and the mutants were expressed in Escherichia coli cells as His-tagged fusion proteins and examined for enzyme characteristics. Tk-PTP possessed two unique features that were not found in eucaryal and bacterial counterparts. First, the recombinant Tk-PTP showed the phosphatase activity not only for the phosphotyrosine but also phosphoserine. Second, the conserved Asp (Asp-63), which was considered to be a critical residue, was not involved in catalysis. In order to know a specific substrate for Tk-PTP, C93S mutant was used to trap substrate protein. Proteins of 120, 60 and 53 kDa were isolated specifically from KODI cell lysates by affinity chromatography with Tk-PTP-C93S. It is suggested that these proteins are tyrosine-phosphorylated substrates of Tk-PTP.