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Literature summary for 3.4.21.91 extracted from

  • Woestenenk, E.; Agback, P.; Unnerstale, S.; Henderson, I.; Agback, T.
    Co-refolding of a functional complex of Dengue NS3 protease and NS2B co-factor domain and backbone resonance assignment by solution NMR (2017), Protein Expr. Purif., 140, 16-27 .
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
separate recombinant expression of N-terminally His-tagged DENV virus 2 NS2B and NS3pro in Escherichia coli in inclusion bodies Dengue virus

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant linked NS2B-NS3pro, X-ray diffraction structure determination and analysis at 1.6 A resolution Dengue virus

Protein Variants

Protein Variants Comment Organism
additional information separate expression of dengue virus NS3 protease and its NS2B cofactor domain and efficient co-refolding to form a stable complex, method evaluation, overview. This straightforward and robust method allows for separate isotope labeling of the two proteins, facilitating analysis by NMR spectroscopy, detailed overview. Unlinked NS2B-NS3pro behaves better in NMR spectroscopy than linked NS2B-NS3pro, which has resulted in the backbone resonance assignment of the unlinked NS2B-NS3 complex bound to a peptidic boronic acid inhibitor Dengue virus

Inhibitors

Inhibitors Comment Organism Structure
2,6-difluoro-benzoyl-Nle-Lys-Arg-Arg-CF3-ketone
-
Dengue virus
benzoyl-Nle-Lys-Arg-Arg-B(OH)2 dynamics of NS2B and NS3pro in the presence of the peptidic inhibitor Dengue virus
additional information unlinked NS2B-NS3 complex bound to a peptidic boronic acid inhibitor Dengue virus

Organism

Organism UniProt Comment Textmining
Dengue virus
-
DENV2
-

Purification (Commentary)

Purification (Comment) Organism
recombinant separately expressed His-tagged NS2B and NS3pro from Escherichia coli, refolded and forming the active complex, by nickel affinity chromatography, tag cleavage by thrombin and/or TEV protease, and another step of nickel affinity chromatography, and gel filtration Dengue virus

Renatured (Commentary)

Renatured (Comment) Organism
recombinant separately expressed His-tagged NS2B and NS3pro from Escherichia coli are co-refolded by one-step dialysis overnight at 4°C in a 2:1 M NS2B:NS3pro ratio to maximize formation of the active complex. The refolding buffer is 25 mM Tris, pH 8.5 (set at 4°C), 5% glycerol, and 100 mM NaCl Dengue virus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
benzoyl-Nle-Lys-Arg-Arg-7-amido-4-methylcoumarin + H2O
-
Dengue virus benzoyl-Nle-Lys-Arg-Arg + 7-amino-4-methylcoumarin
-
?

Synonyms

Synonyms Comment Organism
Dengue NS3 protease
-
Dengue virus
NS2B-NS3 complex
-
Dengue virus
NS2B-NS3pro
-
Dengue virus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.4
-
assay at Dengue virus

Cofactor

Cofactor Comment Organism Structure
NS2B cofactor essential Dengue virus

IC50 Value

IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
0.000019
-
pH 7.4, temperature not specified in the publication, versus the linked NS2B-NS3 complex Dengue virus 2,6-difluoro-benzoyl-Nle-Lys-Arg-Arg-CF3-ketone
0.000038
-
pH 7.4, temperature not specified in the publication, versus the linked NS2B-NS3 complex Dengue virus benzoyl-Nle-Lys-Arg-Arg-B(OH)2