• 제목/요약/키워드: Dual purification system

검색결과 7건 처리시간 0.023초

Expression and Purification of Human Farnesoid X Receptor-Ligand Binding Domain as Soluble Form Using a Dual Cistronic Expression Vector

  • Kang, Hyun;Ye, Micheal B.;Bahk, Young Yil
    • Journal of Microbiology and Biotechnology
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    • 제23권3호
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    • pp.322-328
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    • 2013
  • In this study, we show the expression and purification of the human recombinant farnesoid X receptor (FXR)- ligand binding domain (LBD) protein in E. coli using a double cistronic vector, pACYCDuet-1, as a soluble form. We describe here the expression and characterization of a biologically active $FXR-LBD_{(248-476)}$. When expressed in the influence of bacterial promoters ($P_{T7}$ and $P_{Tac}$) of the single cistronic expression vectors, the human recombinant $FXR-LBD_{(248-476)}$ was found to be totally insoluble. However, by using a double cistronic expression vector, we were able to obtain the human recombinant $FXR-LBD_{(248-476)}$ in a soluble form. To allow for biological activities, we have subcloned into the pACYCDuet-1 vector, expressed in E. coli cells at some optimized conditions, and purified and characterized the human recombinant active $FXR-LBD_{(248-476)}$ proteins using the fluorescence polarization assay. This suggests that the expression of FXR-LBD in a double cistronic vector improves its solubility and probably assists its correct folding for the biologically active form of the proteins. We suggest that this may represent a new approach to high expression of other nuclear receptors and may be useful as well for other classes of heterodimeric protein partners.

Effects of Co-Expression of Liver X Receptor β-Ligand Binding Domain with its Partner, Retinoid X Receptor α-Ligand Binding Domain, on their Solubility and Biological Activity in Escherichia coli

  • Kang, Hyun
    • Journal of Microbiology and Biotechnology
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    • 제25권2호
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    • pp.247-254
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    • 2015
  • In this presentation, I describe the expression and purification of the recombinant liver X receptor β-ligand binding domain proteins in E. coli using a commercially available double cistronic vector, pACYCDuet-1, to express the receptor heterodimer in a single cell as the soluble form. I describe here the expression and characterization of a biologically active heterodimer composed of the liver X receptor β-ligand binding domain and retinoid X receptor α-ligand binding domain. Although many of these proteins were previously seen to be produced in E. coli as insoluble aggregates or "inclusion bodies", I show here that as a form of heterodimer they can be made in soluble forms that are biologically active. This suggests that co-expression of the liver X receptor β-ligand binding domain with its binding partner improves the solubility of the complex and probably assists in their correct folding, thereby functioning as a type of molecular chaperone.

제주특별자치도 상수도 기후위기 적응대책 연구 (A study on adaptation measures to climate crisis for water supply system of Jeju Special Self-Governing Province)

  • 김진근
    • 상하수도학회지
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    • 제37권6호
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    • pp.447-456
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    • 2023
  • Risk assessment on Jeju Special Self-Governing Province(JSSGP)'s water supply facilities and establishment of adaptation measures for climate crisis factors were implemented. JSSGP's vulnerability to the climate crisis was high in the order of drought, heat wave, heavy rain and strong wind. As a drought adaptation measure, policies of water saving and revenue water ratio improvement were considered. As for the heat wave adaptation measure, the introduction of an advanced water treatment process was suggested in response to the increase of algae cell number which resulting in taste and odor problem. As for heavy rain adaptation measures, the installation and operation of automatic coagulant injection devices for water purification plants that take turbid surface water were proposed. As a measure to adapt to strong winds, stabilization of power supply such as installation of dual power line was proposed in preparation for power outages. It is expected that water facilities will be able to supply high-quality tap water to customers even under extreme climate conditions without interruption through risk assessment for climate crisis factors and active implementation of adaptation measures.

핵연료 시험용 노내조사시험설비의 설계 현황 (The Design Status of the Irradiation Facility for Fuel Test)

  • 박국남;심봉식;안성호;유성연
    • 대한설비공학회:학술대회논문집
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    • 대한설비공학회 2007년도 동계학술발표대회 논문집
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    • pp.310-315
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    • 2007
  • The FTL has been developed to be able to irradiate test fuels at the irradiation hole(IR1 hole) by considering its utility and user's irradiation requirements. FTL consists of In-Pile Test Section (IPS) and Out-of-Pile System (OPS). Test condition in IPS such as pressure, temperature and the water quality, can be controlled by OPS. For safety assurance IPS is designed to have dual stainless steel pressure vessel and OPS is composed of main cooling water system, emergency cooling water system, LMP(letdown, make-up, purification) system, etc. FTL Conceptual design was set up in 2001, basic design had completed including a design requirement, basic piping & instrument diagram (P&ID), and the detail design in 2004. In 2005, the development team carried out purchase and manufacture hardware and make a contract for construction work. FTL construction work began on August, 2006 and ended on March, 2007. After FTL development which is expected to be finished by 2008, FTL will be used for the irradiation test of the new PWR-type fuel and can maximize the usage of HANARO.

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Bombyx mori Nucleopolyhedrovirus Bacmid Enabling Rapid Generation of Recombinant Virus by In Vitro Transposition

  • Tao, Xue Ying;Choi, Jae Young;Kim, Yang-Su;Lee, Seok Hee;An, Saes Byeol;Pang, Ying;Kim, Jong Hoon;Kim, Woo Jin;Je, Yeon Ho
    • Journal of Microbiology and Biotechnology
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    • 제25권3호
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    • pp.386-392
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    • 2015
  • A novel recombinant bacmid, bEasyBm, that enables the easy and fast generation of pure recombinant baculovirus without any purification step was constructed. In bEasyBm, attR recombination sites were introduced to facilitate the generation of a recombinant viral genome by in vitro transposition. Moreover, the extracellular RNase gene from Bacillus amyloliquefaciens, barnase, was expressed under the control of the Cotesia plutellae bracovirus early promoter to negatively select against the nonrecombinant background. The bEasyBm bacmid could only replicate in host insect cells when the barnase gene was replaced with the gene of interest by in vitro transposition. When bEasyBm was transposed with pDualBac-EGFP, the resulting recombinant virus, EasyBm-EGFP, showed high levels of EGFP expression efficiency compared with that of non-purified recombinant virus BmGOZA-EGFP, which was constructed using the bBmGOZA system. In addition, nonrecombinant backgrounds were not detected in unpurified EasyBm-EGFP stocks. Based on these results, a high-throughput system for the generation of multiple recombinant viruses at a time was established.