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Purification and conformational changes of bovine PEGylated β-lactoglobulin related to antigenicity
Junzhen Zhong1, Xiaofei Cai1, Chengmei Liu1
1State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China.
β-Lactoglobulin (β-LG) was conjugated with monomethoxy polyethylene glycol-succinimidyl carbonates (mPEG-SC, 20 kDa) to investigate the relationship between the antigenicity and conformational changes of β-LG. The effect of molar ratio of protein to mPEG-SC (1:3-1:6), pH (6-8) and time (4-24h) on the antigenicity of β-LG was investigated. The lowest antigenicity of β-LG was observed at the molar ratio of 1:3, pH 7.0, and reaction time for 8h, which was 70% lower than that of control β-LG. At the optimal modification conditions, it was indicated that two fractions obtained after purification showed the tense and single band on the SDS-PAGE at the position of approximate 78 kDa and 58 kDa, which corresponded to the tri- and di-PEGylated conjugate, respectively. As conjugated number of mPEG-SC with β-LG increased, the quenching of fluorescence and the content of β-strands were increased gradually, which may contribute to the decrease of antigenicity from two aspects.
β-Lactoglobulin (β-LG) was conjugated with monomethoxy polyethylene glycol-succinimidyl carbonates (mPEG-SC, 20 kDa) to investigate the relationship between the antigenicity and conformational changes of β-LG. The effect of molar ratio of protein to mPEG-SC (1:3-1:6), pH (6-8) and time (4-24h) on the antigenicity of β-LG was investigated. The lowest antigenicity of β-LG was observed at the molar ratio of 1:3, pH 7.0, and reaction time for 8h, which was 70% lower than that of control β-LG. At the optimal modification conditions, it was indicated that two fractions obtained after purification showed the tense and single band on the SDS-PAGE at the position of approximate 78 kDa and 58 kDa, which corresponded to the tri- and di-PEGylated conjugate, respectively. As conjugated number of mPEG-SC with β-LG increased, the quenching of fluorescence and the content of β-strands were increased gradually, which may contribute to the decrease of antigenicity from two aspects.
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