Targeted Diminution of the Immunogenicity of the Red Fluorescent Protein TagRFP

Introduction. Estimation of the response of organs and systems of the body to the new medicinal substances action is an important step in drug development. Therefore, visualization tools for these processes, such as fluorescent proteins, should be minimally involved in the biochemistry of the cell or organism. In particular, GFP-like fluorescent proteins used as genetically encoded sensors in experiments on laboratory animals can be immunogenic, just like other proteins. This can lead to unreliable experimental assessment of the organism’s reaction to potential drugs and therapies. The aim of this work was to create new mutants of the red fluorescent protein TagRFP with reduced immunogenicity in BALB/c mice compared to the original TagRFP. Methods. Potential epitopes for BALB/c mice were selected in silico within the primary sequence of the TagRFP protein, and for those that least affect folding and fluorophore formation, amino acid substitutions to alanine were analyzed. At the selected positions, Y225A and L228A substitutions were made via site-directed mutagenesis, and the genes were cloned into a prokaryotic plasmid. Using the above plasmids, the new mutant proteins were expressed in E. coli BL21(DE3), isolated, and purified. BALB/c mice were immunized with the obtained proteins, and the humoral immune response was evaluated using enzyme-linked immunosorbent assay (ELISA). Results. It has been shown that the physicochemical properties of the mutant forms TagRFP-Y225A and TagRFP-L228A have no significant differences compared with the parent protein. After immunization with the TagRFP-L228A mutant, the binding of mouse serum to TagRFP-WT decreased compared to serum binding after immunization with TagRFP-WT, for both native and denatured proteins. At the same time, when mice were immunized with the TagRFP-Y225A mutant, this occurred only for the denatured protein. This is probably due to the fact that Y225 is located immediately after β-barrel, and in the native form, changes in these epitopes are “shielded” from recognition by the immune system. Conclusions. Two mutant proteins, TagRFP-Y225A and TagRFP-L228A, were obtained based on the red fluorescent protein TagRFP, with reduced immunogenicity for BALB/c mice.