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.
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The data underlying this article are available in the article and also from the corresponding author upon request.
Abbreviations
- BLG:
-
β-Lactoglobulin
- wt:
-
Wild-type
- SDS–PAGE:
-
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
- PBS:
-
Phosphate-buffered saline
- mAb:
-
Monoclonal antibody
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Acknowledgements
This work was supported in part by JSPS KAKENHI Grant Number JP17580107.
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TY, MT: Conceptualization, Funding acquisition, Writing manuscript, KY: Conceptualization, Data curation, Writing manuscript, CK, AS, FY, NT, RK: Data curation, Writing manuscript, MH: Conceptualization, Writing manuscript, Funding acquisition.
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Yoshida, T., Kume, C., Sachi, A. et al. Reduced immunogenicity of β-lactoglobulin by single amino acid substitution. Cytotechnology 74, 623–634 (2022). https://doi.org/10.1007/s10616-022-00549-9
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DOI: https://doi.org/10.1007/s10616-022-00549-9