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The Dengue Virus Protease Complex NS2B/NS3 - A Novel TargetFor The Structure-Based Design of Improved Antiviral Agents

หน่วยงาน สำนักงานกองทุนสนับสนุนการวิจัย

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ชื่อเรื่อง : The Dengue Virus Protease Complex NS2B/NS3 - A Novel TargetFor The Structure-Based Design of Improved Antiviral Agents
นักวิจัย : Gerd Katzenmeier
คำค้น : assay , cofactor , Dengue virus , expression , fluorescence , HPLC , NS2B , NS3 , polyprotein , purification , serine protease , substrate
หน่วยงาน : สำนักงานกองทุนสนับสนุนการวิจัย
ผู้ร่วมงาน : -
ปีพิมพ์ : 2546
อ้างอิง : http://elibrary.trf.or.th/project_content.asp?PJID=BRG4180005 , http://research.trf.or.th/node/1918
ที่มา : -
ความเชี่ยวชาญ : -
ความสัมพันธ์ : -
ขอบเขตของเนื้อหา : -
บทคัดย่อ/คำอธิบาย :

The objective of this project is the detailed characterization of the dengue virus protease complex NS2B/NS3 by molecular biological and biochemical methods in order to analyse structure-activity relationships of the enzyme with a view to the rationale- based design of antiviral compounds. During the course of this project, we have constructed recombinant clones of the NS2B/NS3 protease components in Escherichia coli which over-express the target proteins and we have developed biochemical methods for their purification and refolding. The dengue virus proteins NS2B, NS3(pro) and NS2B/NS3 from dengue virus type 2 were expressed as fusion proteins containing hexahistidine affinity tags and were purified to near-homogeneity by Ni - affinity metal chelate chromatography. Several methods for the refolding of the NS3 protease were explored and stepwise dialysis was used to renature the enzyme to its active conformation. A major part of the project was the development of an assay system to monitor NS3 proteolytic activity. A number of methods were evaluated and currently a fluorometric assay with short peptides and a HPLC-based assay using dansylated 12mer peptide substrates which mimick authentic dengue virus polyprotein cleavage sites is used for the analysis of NS3 protease biochemical reaction parameters. We have shown that all peptides encompassing native polyprotein cleavage sites are specifically cleaved by the NS3 enzyme. The assay is suitable for the analysis of substrate requirements, structural determinants of cleavage activity and -specificity and for the evaluation of potential inhibitors. An alternative expression system for the NS3 protein in the yeast Pichia pastoris was explored and we could show that NS3(pro) fused to the yeast alpha-factor was expressed at high levels albeit not secreted into the culture medium. A recombinant fusion protein containing the hydrophilic subdomain of NS2B fused to NS3(pro) was expressed in E. coli and we have demonstrated autoproteolytic processing at the NS2B- NS3 site after purification and refolding. This experimental system will be useful to further characterize the mechanism of cofactor activation of the NS3 protease. The project is now equipped with a number of useful tools and techniques to address the biochemical research problems associated with a structural and functional characterization of the DEN NS3 protease.

บรรณานุกรม :
Gerd Katzenmeier . (2546). The Dengue Virus Protease Complex NS2B/NS3 - A Novel TargetFor The Structure-Based Design of Improved Antiviral Agents.
    กรุงเทพมหานคร : สำนักงานกองทุนสนับสนุนการวิจัย.
Gerd Katzenmeier . 2546. "The Dengue Virus Protease Complex NS2B/NS3 - A Novel TargetFor The Structure-Based Design of Improved Antiviral Agents".
    กรุงเทพมหานคร : สำนักงานกองทุนสนับสนุนการวิจัย.
Gerd Katzenmeier . "The Dengue Virus Protease Complex NS2B/NS3 - A Novel TargetFor The Structure-Based Design of Improved Antiviral Agents."
    กรุงเทพมหานคร : สำนักงานกองทุนสนับสนุนการวิจัย, 2546. Print.
Gerd Katzenmeier . The Dengue Virus Protease Complex NS2B/NS3 - A Novel TargetFor The Structure-Based Design of Improved Antiviral Agents. กรุงเทพมหานคร : สำนักงานกองทุนสนับสนุนการวิจัย; 2546.