Fig. 1

Design and analysis of hybrid flagellin protein. a Schematic of the construction and design of hybrid flagellin (hFliC). The D3 domain from the native FliC protein was deleted (residues 185 to 285). DENV2 E plus GS linkers flanking the termini were inserted to replace the D3 domain of FliC. The final gene product was cloned into the pENTR plasmid for baculovirus expression of the hybrid protein. b Protein expressed by baculovirus expression system was checked for purity and anticipated molecular weight (~85 kDa) using both Coomassie stain (left panel, which labels all proteins) and Western blot (right panel). In the Western blot, protein was probed with an antibody against the His-tag under denaturing conditions. Major bands from both blots indicate appropriate band size, however some degradation products were present in Coomassie gel; by densitometry the predicted hFliC band comprised approximately 15 % of total protein. The double band in the Western blot suggests a proteolytic cleavage very near the N-terminus. c Circular dichroism (CD) was used to assess secondary structural feature of the hFliC protein. CD spectra of FliC (blue curve) exhibit a characteristic α-helical region, while DENV2 spectra (green curve) exhibit a large β-sheet and coil regions. hFliC CD spectra (red curve) was essentially a hybrid of the two proteins