Enhancement of antigen-specific humoral immune responses and protein solubility through conjugation of bacterial flagellin, Vibrio vulnificus FlaB, to the N-terminus of porcine epidemic diarrhea virus surface protein antigen S0

Oh, Seo-ho; Kim Cho, Young-Saeng; Lee, Ho-Bin; Lee, Sang-Mok; Kim, Whee-Soo; Hong, Liang; Cho, Chong-Su; Choi, Yun-Jaie; Kang, Sang-Kee

Interestingly, despite their similar molecular weights, the total amount of F-S0 expression was approximately 6.2-and 1.5-times higher than S0-F in the presence or absence of tig, respectively (Fig. 1) . This difference in protein levels suggests that the N-terminal flagellin sequence may offer efficient translation initiation, resulting in increased production of F-S0. In fact, it is generally accepted that fusion of a solubility enhancer to the N-terminus of a target protein can enable efficient translation initiation, which makes it a more favorable choice for soluble expression than the C-terminal fusion [28] . The conjugation of flagellin to the C-terminus of S0 (S0-F) improved soluble expression in comparison to the N-terminal conjugation (F-S0) or S0 alone in the absence of tig ( Fig. 1A and C) . tig enhances the solubility of recombinant protein through by assisting with the protein folding process. Proteins that are prone to aggregation, including the S1D and COE region of PEDV's spike protein, which are otherwise totally insoluble, have been expressed as soluble proteins with the assistance of tig [9] . In contrast, the condition lacking tig had a relatively lower capacity to fold recombinant protein correctly and make soluble protein. Therefore, rapid translation of F-S0 in the absence of tig is thought to produce IBs rather than soluble protein in spite of its total protein expression level being 1.5-times higher than the S0-F protein level.