Effect of PEGylation of β-Lactoglobulin at Carboxyl Residues on Its Structure and Antigenicity

doi:10.7506/spkx1002-6630-20200803-039

Abstract

Abstract: The effect of PEGylation and PEGylation degree on the antigenicity and structure of β-lactoglobulin were investigated by covalent binding of PEG to the carboxyl group of β-lactoglobulin. The results showed that the total modification rate was 82.91%. The PEGylation products were separated and purified using an SP Sepharose Fast Flow cation exchange column into two major fractions with purity of 99.66% and 98.61%. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) the relative molecular weights of the products identified as 23.3 and 28.6 kDa, respectively, indicating that they were mono-PEG-β-LG and di-PEG-β-LG. The half-maximal inhibitory concentration (IC50) values of β-LG, mono-PEG-β-LG and di-PEG-β-LG were 1.90, 2.47 and 10.41 μg/mL, respectively as determined by indirect competitive enzyme-linked immunosorbent assay (IC-ELISA). The IC50 values of mono-PEG-β-LG and di-PEG-β-LG were 1.30 and 5.48 times higher than that of β-LG, respectively, which indicated that the antigenicity of β-LG could be significantly reduced by increasing the PEGylation degree. The free sulfhydryl contents of mono-PEG-β-LG and di-PEG-β-LG were 42.70 and 37.97 μmol/g, respectively, indicating that as the PEGylation degree increased, the masking effect increased. Surface hydrophobicity analysis and intrinsic fluorescence analysis showed changes in tertiary structure only for mono-PEG-β-LG, and in both secondary and tertiary structure for di-PEG-β-LG, indicating that the spatial structure changes of β-LG after PEGylation might be the cause of the destruction of its conformational epitope and the decrease of its antigenicity. The masking of PEG molecules might be another reason for the reduced antigenicity of products. The covalent PEGylation of the carboxyl group of β-LG could significantly reduce its antigenicity, and the antigenicity decreased significantly with the increase of PEGylation degree.

Key words: β-lactoglobulin; polyethylene glycol modification; antigenicity; structure change

CLC Number:

TS201.4

LUO Shunjing, JI Li, XIONG Shaobai, ZHONG Junzhen, ZHU Xiaoming, JIANG Xinlin, LIU Chengmei. Effect of PEGylation of β-Lactoglobulin at Carboxyl Residues on Its Structure and Antigenicity[J]. FOOD SCIENCE, 2021, 42(24): 16-23.

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Effect of PEGylation of β-Lactoglobulin at Carboxyl Residues on Its Structure and Antigenicity

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