Reduced immunogenicity of β-lactoglobulin by single amino acid substitution

Tadashi Yoshida; Chisato Kume; Asako Sachi; Fumiko Yuyama; Naoko Tomiyama; Rina Kodama; Kiyoshi Yamada; Mamoru Totsuka; Makoto Hattori

doi:10.1007/s10616-022-00549-9

Reduced immunogenicity of β-lactoglobulin by single amino acid substitution

Cytotechnology. 2022 Dec;74(6):623-634. doi: 10.1007/s10616-022-00549-9. Epub 2022 Sep 30.

Authors

Tadashi Yoshida¹, Chisato Kume¹, Asako Sachi¹, Fumiko Yuyama¹, Naoko Tomiyama¹, Rina Kodama¹, Kiyoshi Yamada², Mamoru Totsuka², Makoto Hattori¹

Affiliations

¹ Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu City, Tokyo, 183-8509 Japan.
² Department of Applied Biological Chemistry, The University of Tokyo, Tokyo, 113-8657 Japan.

Abstract

To reduce the immunogenicity of β-lactoglobulin (BLG), we prepared single amino acid substituted recombinant BLG mutants (BLG/P126A, BLG/V128D and BLG/D129A) in the methylotrophic yeast Pichia Pastris by fusion of the cDNA to the sequence coding for the α-factor signal peptide from Saccharomyces cerevisiae. Isoelectric points of single amino acid substituted BLGs were lower than that of native BLG. CD spectra indicated that the secondary structure of BLG had maintained native structure in single amino acid substituted BLGs. Fluorescence studies indicated that the conformation around Trp had not changed in single amino acid substituted BLGs. Anti-BLG antibody response was evaluated after immunization to C57BL/6 mice. Antibody response was reduced after immunization with BLG/P126A, BLG/V128D and BLG/D129A. And novel immunogenicity was not observed in the experiments. T cell proliferative response was evaluated in C57BL/6 mice, and it was clarified that BLG mutants also showed low response. Methods employed in this study was considered to be very effective to reduce immunogenicity of BLG.

Keywords: Protein engineering; Reduced immunogenicity; Single amino acid substitution; β-Lactoglobulin.

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