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. 2008 Jun 11;82(16):8085–8093. doi: 10.1128/JVI.00693-08

FIG. 5.

FIG. 5.

Sapovirus NS6-7 protein processes the cleavage site NS5-NS6 in cis. The cDNA clones pIR-SAP-ΔτNS5NS6/tNS6-7 and pIR-SAP-ΔτNS4NS5/tNS6-7 were used for expression in Escherichia coli Rosetta(DE3) cells, yielding proteins HA-[NS5/NS6]-NS6-7-His and HA-[NS4/NS5]-NS6-7-His, respectively. Protein HA-[NS5/NS6]-NS6-7-His bears at the N terminus the authentic NS5-NS6 cleavage site (PQQHYEEE1053/A) fused to an HA tag, and protein HA-[NS4/NS5]-NS6-7-His bears at the N terminus the authentic NS4-NS5 cleavage site (TRGSRTEE939AKGKTKHG) fused to an HA tag. The proteins were expressed in E. coli, the cultures were harvested 8 h after induction, and the pellets were diluted 1:25, 1:50, 1:75, and 1:100, analyzed by SDS-PAGE, and visualized by Western blotting using anti-HA or anti-His antibodies. Western blot analysis of the eluted proteins diluted to 1:25, 1:50, 1:75, and 1:100 is shown. (A) Detection of proteins HA-[NS5/NS6]-NS6-7-His and HA-[NS4/NS5]-NS6-7-His by anti-His antibody, as indicated. (B) Detection of proteins HA-[NS5/NS6]-NS6-7-His and HA-[NS4/NS5]-NS6-7-His by anti-HA antibody, as indicated.