A systematic mutational analysis identifies a 5‐residue proline tag that enhances the in vivo immunogenicity of a non‐immunogenic model protein

Poor immunogenicity of small proteins is a major hurdle in developing vaccines or producing antibodies for biopharmaceutical usage. Here, we systematically analyzed the effects of 10 solubility controlling peptide tags (SCP‐tags) on the immunogenicity of a non‐immunogenic model protein, bovine pancreatic trypsin inhibitor (BPTI‐19A; 6 kDa). CD, fluorescence, DLS, SLS, and AUC measurements indicated that the SCP‐tags did not change the secondary structure content nor the tertiary structures of the protein nor its monomeric state. ELISA results indicated that the 5‐proline (C5P) and 5‐arginine (C5R) tags unexpectedly increased the IgG level of BPTI‐19A by 240‐ and 73‐fold, respectively, suggesting that non‐oligomerizing SCP‐tags may provide a novel method for increasing the immunogenicity of a protein in a highly specific manner.