Recombinant dengue type 1 virus NS5 protein expressed in Escherichia coli exhibits RNA-dependent RNA polymerase activity.
The complete nonstructural NS5 gene of dengue type 1 virus, Singapore strain S275/90 (D1-S275/90) was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein (126 kDa). The GST-NS5 fusion protein was purified and the recombinant NS5 protein released from the fusion protein by thrombin cleavage. The recombinant NS5 had a predicted molecular weight of 100 kDa and reacted with antiserum against D1-S275/90 virus in Western blot analysis. The purified recombinant NS5 protein possessed RNA-dependent RNA polymerase activity which was inhibited (>99%) by antibodies against the recombinant NS5 protein. The polymerase product was shown to be a negative-stranded RNA molecule, of template size, which forms a double-stranded complex with the template RNA.
Tan
B H
BH
Institute of Molecular and Cell Biology, National University of Singapore.
Fu
J
J
Sugrue
R J
RJ
Yap
E H
EH
Chan
Y C
YC
Tan
Y H
YH
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D003716:Dengue Virus / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; C096041:NS5 protein, flavivirus; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase