Abstract: In order to investigate the effect of different covalent modification sites on the antigenicity of β-lactoglobulin (β-LG), β-LG was modified by using cysteine-specific PEGylation at the thiol group of Cys121. Using one-factor-at-a-time method, the optimal modification conditions were determined as follows: β-LG and mPEG-MAL (molar ratio, 1:30) were dissolved together in 0.1 mol/L phosphate buffer containing 0.3 mol/L tris-(2-carboxyethyl) phosphine (TCEP), 25% (V/V) ethanol and 2 mmol/L ethylenediaminetetraacetic acid (EDTA) at pH 7.0 and incubated at 4 ℃ for 24 h, yielding a maximum modification efficiency of 64.1%. The resulting product was purified to a purity of 94%. The free sulfhydryl content of β-LG decreased from 52.3 to 0.3 μmol/g after the site-specific PEGylation. It was demonstrated that mPEG-MAL was specifically conjugated with β-LG at the thiol group of Cys121. The antigenicity of β-LG increased by 25.9% after the cysteine-specific PEGylation, contrary to results previously obtained by modification at other amino acid positions. Accordingly, site-specific PEGylation can allow effective regulation of β-LG antigenicity.

Key words: β-lactoglobulin; polyethylene glycol; covalent modification; site-specific PEGylation; antigenicity