A novel method for synthetic vaccine construction based on protein assembly

Liu, Zhida; Zhou, Hang; Wang, Wenjun; Tan, Wenjie; Fu, Yang-Xin; Zhu, Mingzhao

Optimization of SpyCatcher. The current SpyTag/SpyCatcher system consists of a 13 aa SpyTag and a 138 aa SpyCatcher. To further simplify this system for engineering purposes and to minimize its immunogenicity, we tried to truncate the SpyCatcher protein while maintaining its conjugation activity. A structural analysis found that aa 53-118 are probably essential for the conjugation activity. An immunogenicity analysis revealed four major immune epitopes at the N-terminus (aa 32-50, aa 57-66) and C-terminus (aa 104-112, aa 121-138) ( Figure S1 ). Considering these together, we performed several truncations, as shown in Figure 1a . The truncation with deletion at the N-terminus (24-47 aa) was named SpyCatcherDN, and the truncation with deletions at both the N-terminus and the C-terminus (24-47 and 121-138 aa) was named SpyCatcherDNC. The truncated SpyCatcher proteins were expressed in Escherichia coli and purified by Ni-NTA chromatography ( Figure 1b) . The full-length and truncated SpyCatcher proteins were then used to immunize C57BL/6 mice. Fourteen days later, antibody levels in the sera were determined by ELISA. As shown in Figure 1c , the antibody levels induced by SpyCatcherDN and SpyCatcherDNC were significantly lower than those induced by full-length SpyCatcher. No significant difference was found between SpyCatcherDN and SpyCatcherDNC. Next, we further tested the efficiency of the binding of the truncated SpyCatcher proteins to the SpyTag fusion protein, aDEC205-SpyTag, which was made by genetic fusion of SpyTag with a single-chain antibody against murine DEC205 (aDEC205) at its C-terminus. As shown in Figures 1d and 1e , there was no significant difference in binding efficiency between SpyCatcherDN and full-length SpyCatcher, consistent with the results of a recent study 24 . However, the binding efficiency of SpyCatcherDNC was obviously lower than that of full-length SpyCatcher. Therefore, SpyCatcherDN was chosen for further studies.