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생화학분자생물학회 (Korean Society for Biochemistry and Molecular Biology)
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In Vitro Determination of Dengue Virus Type 2 NS2B-NS3 Protease Activity with Fluorescent Peptide Substrates

  • Khumthong, Rabuesak (Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus) ;
  • Angsuthanasombat, Chanan (Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus) ;
  • Panyim, Sakol (Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus) ;
  • Katzenmeier, Gerd (Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus)
  • 발행 : 2002.03.31
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초록

The NS2B-NS3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing E. coli by metal chelate affinity chromatography and gel filtration. Enzymatic activity of the refolded NS2B-NS3(pro) protease complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites. The 12mer substrate peptides and the cleavage products could be separated by reversed-phase HPLC, and were identified by UV and fluorescence detection. All of the peptide substrates (representing the DEN polyprotein junction sequences at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites) were cleaved by the recombinant protease NS2B-NS3(pro). No cleavage was observed with an enzymatically inactive S135A mutant of the NS3 protein, or with a modified substrate peptide of the NS3/NS4A polyprotein site that contained a K2093A substitution. Enzymatic activity was dependent on the salt concentration. A 50% decrease of activity was observed in the presence of 0.1M sodium chloride. Our results show that the NS3 protease activity of the refolded NS2B-NS3(pro) protein can be assayed in vitro with high specificity by using cleavage-junction derived peptide substrates.

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참고문헌

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