Fig. 4. Expression of recombinant CuquEndo in 293 cells and SDS-PAGE agarose gel assay. (A) In vitro assay showing the presence of endonuclease activity in supernatant of cells transfected with the CuquEndo-VR2001 construct. No plasmid degradation was observed in the supernatant of 293 cells transfected with an empty VR2001 plasmid (mock control) P, 500 ng of doubled stranded plasmid DN, Ladder, 1 kb Plus DNA ladder. (B) Concentrated 293 cell supernatant (crude) containing the recombinant CuqueEndo was resolved in a NuPAGE gel. After in gel renaturation (see Materials and methods) the NuPAGE gel was exposed to a 1% agarose gel containing 0.54 ng ml^-1 of double stranded plasmid DNA. Degradation of plasmid DNA occurred at the position of the calculated molecular mass of recombinant CuquEndo, as visualized with Ethidium Bromide under UV light. (C) The Coomassie Blue-stained NuPAGE gel (arrow indicates position of CuquEndo). Blue, prestained standard proteins.