In:
Bioscience, Biotechnology, and Biochemistry, Oxford University Press (OUP), Vol. 87, No. 4 ( 2023-03-21), p. 426-433
Abstract:
To reduce the immunogenicity of β-lactoglobulin (BLG), we prepared recombinant BLG which has both site-specific glycosylation and single amino acid substitution (D28N/P126A), and expressed it in the methylotrophic yeast Pichia pastoris by fusion of the cDNA to the sequence coding for the α-factor signal peptide from Saccharomyces cerevisiae. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis indicated that the D28N/P126A was conjugated with a ∼4 kDa high-mannose chain. D28N/P126A retained ∼61% of the retinol-binding activity of BLG. Structural analyses by circular dichroism (CD) spectra, intrinsic fluorescence, and Enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies indicated that the surface structure of BLG was slightly changed by using protein engineering techniques, but D28N/P126A was covered by high-mannose chains and substituted amino acid without substantial disruption of native conformation. Antibody responses to the D28N/P126A considerably reduced in C57BL/6 mice. We conclude that inducing both site-specific glycosylation and single amino acid substitution simultaneously is an effective method to reduce the immunogenicity of BLG.
Type of Medium:
Online Resource
ISSN:
1347-6947
Language:
English
Publisher:
Oxford University Press (OUP)
Publication Date:
2023
detail.hit.zdb_id:
2110940-0
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