Fig. 5

Analysis and expression of the mutated HEV p146 and NcoI-E2s. The same three mutations were introduced into p146 and NcoI-E2s: the second base of the codon 457 (C with A) and the first and second positions of the codon 458 (CC with GA). The predicted mRNA secondary structures near the start codons of the mutated genes shows that the start codon AUG and the SD sequence are exposed from the stable stem-loop structural context (a). The accessibility analysis (b) shows that the SD sequence in the mRNA of the mutated genes requires less thermodynamic energy to be accessible compared to the wild-type genes, indicating that the mutations allowed the exposition of the SD sequence. The mutated proteins were successfully over-expressed in E. coli (BL21) as shown by a 15% SDS-PAGE analysis (c, top) and were reactive against the anti-HEV 4C5 monoclonal antibody as shown by Western blot analysis (c, bottom)