Analysis of bacterially purified human endonuclease. (A) Schematic of a full-length L1, which contains a 5′ untranslated region (UTR) followed by an ORF1 sequence, an intergenic region, an ORF2 sequence, and a 3′ UTR. The EN region of the ORF2 sequence subcloned to generate the purified ORF2p endonuclease (EN, 1-239aa) is indicated with a dashed box. (B) (Left panel) Coomassie stain of SDS-PAGE gel. Ladder (L), clarified lysate from bacteria expressing ORF2p endonuclease (CL), and final purified elution (PE) are shown; 500 ng of protein was loaded in each lane. (Middle panel) Western blot analysis of 500 ng of CL and PE with HIS-tag specific antibodies. The ORF2p endonuclease used in this study has a HIS-tag fused to its N-terminus (expected size of the His EN protein is 29 kilodaltons, kDa). (Right panel) Western blot analysis of 500 ng of CL and PE with a custom anti-human ORF2p monoclonal antibody. Molecular markers on the right, 10 to 250 kDa.