Effect Of PEGylation Of β-lactoglobulin At Carboxyl Residues On Its Antigenicity And Structure

β-lactoglobulin(β-LG)is one of the main components of milk allergy,and it is necessary to reduce its allergenicity for people with allergies.The use of PEGylation to reduce the allergenicity of β-LG is a hotspot in the international dairy processing research.The results of PEGylation at different groups of β-LG showed that different binding sites had different effects on the structure and antigenicity of β-LG.In order to comprehensively explore the effects of different PEG binding sites and PEGylation degree on the antigenicity and structure of β-LG,5 kDa methoxy polyethylene glycol hydrazide(m PEG-Hz,5 kDa)was used to modify the carboxyl groups of β-LG,and two PEGylation products with different PEGylation degrees were obtained.Then the two PEGylation products were isolated,purified and identified.The structure,antigenicity and binding sites of the PEGylation products were analyzed.The effects of binding sites and PEGylation degree on the structure and antigenicity were explored,and the machanism of antigenicity changes was premilinarily discussed,which provided a theoretical basis for regulating the antigenicity of β-LG.The carboxyl groups of β-LG were covalently modified with 5 kDa m PEG-Hz,and the PEGylation conditions were optimized.The reaction p H,molar ratio of β-LG to PEG,β-LG to EDC,and EDC to sulfo-NHS were optimized by SDS-PAGE and gel filtration chromatography with the PEGylation rates as the index.The results showed that the above four factors had great influence on the modification rates.At the conditions of p H 4.0,molar ratio of β-LG to PEG 1:20,β-LG to EDC 1:50,EDC to sulfo-NHS 5:1,the total modification rate was the highest,which was 82.91%.The cation exchange chromatography was used to isolate and purify,and two elution peaks of products were obtained,with the purity 99.66% and 98.61%respectively.The results of SDS-PAGE showed that the apparent molecular eights of two products were between 26-34 kDa and 34-43 kDa,respectively.The actual molecular weights of β-LG and the two products were 18.3 kDa,23.3 kDa and 28.6kDa,which proves that the two PEGylated products were mono-PEG-β-LG and diPEG-β-LG,respectively.The structure analysis showed that after PEGylation,the content of free sulfhydryl was significantly reduced,the fluorescence intensity decreased,and the surface hydrophobicity increased,showing that the tertiary structure was changed after PEGylation.There were no significant changes in the secondary structure of mono-PEG-β-LG,while the di-PEG-β-LG had significant changes in the secondary structure,which showed that the α-helix content decreased,the random coil content increased.The half maximal inhibitory concentration(IC50)of β-LG was rised from1.90 μg/m L to 2.47 μg/m L(mono-PEG-β-LG)and 10.41 μg/m L(di-PEG-β-LG)respectively,showing that the antigenicity was significantly reduced.The higher the PEGylation degree,the lower the antigenicity.The PEGylation sites analysis showed that the mostly likely binding site for mono-PEG-β-LG was Ile162,di-PEG-β-LG was most likely to bind the first PEG at Ile162 and the second PEG to one of Asp127,Glu130 and Glu134.The antigenicity of β-LG can be reduced significantly by PEGylation at carboxyl groups.The shielding effect of PEG molecule and the destruction of conformational epitopes caused by the change of β-LG tertiary structure were the two reasons for the reduction of the antigenicity of PEGylation products.The higher the degree of PEGylation,the more obvious the masking and the destruction of conformational epitopes,the lower the antigenicity of the PEGylation products.Combined with previous studies,it can be found that both binding sites of PEG molecules and PEGylation degree had great influence on the antigenicity of β-LG.