Current data revealed removal of 6xHis-tag appeared not to affect the CFA/I/II/IV MEFA at protein expression, protein structure and stability, and immunogenicity. Data showed the tag-less and 6xHis-tagged CFA/I/II/IV MEFA proteins were expressed and extracted at the same yield (about 150 mg per liter culture medium) and purity (greater than 95%, based on PAGE Coomassie blue staining and mass spectrophotometer). Molecular dynamics simulation suggested a stable structure for the tag-less or the 6xHis-tagged MEFA (data not shown for the 6xHis-tagged MEFA). This tag-less CFA/I/II/IV MEFA showed equally or more immunogenic than the his-tagged CFA/I/II/IV MEFA (Figure 7), although the enhanced immunogenicity exhibited by the tag-less CFA/I/II/IV MEFA could be resulted from dmLT adjuvant, since dmLT was demonstrated to be equally or more effective (compared to Freund’s adjuvant) to immunoregulate parenterally immunized ETEC antigens . The 6xHis-tag typically sticks out at protein surface (designed for nickel ion attachment during protein purification process) and may affect the exposure of the adjacent epitopes, through such negative effect could be limited presumably because the his-tag consists of only six histidines and eleven other residues of expression vector pET28α. Data from the current study showed the tag-less CFA/I/II/IV MEFA displayed immunogenicity to each representing epitope computationally and empirically.