Effect Of Site-specific PEGylation Of β-lactoglobulin At Cysteine Residue On Its Conformational Changes And Antigenicity

Cow’s milk allergy is one of the food safety problems that are concerned worldwide.Among more than 30 kinds of potential bovine milk antigenicity proteins,β-lactoglobulin(β-LG)is recognized as the most important allergen in bovine whey.Research on the antigenicity of β-LG is very extensive.The use of covalent modification to investigate β-LG antigenicity has become a hot spot in international dairy processing technology.However,most of the commonly used covalent modification methods are multi-site random modification,which cannot accurately regulate the antigenicity of β-LG.In this study,the site-specific polyethylene glycol(PEG)modification method was adopted to realize effective control on the antigenicity of β-LG.Compared with the traditional random modification method,the site-specific PEGylation can be used to modify specific groups or sites of proteins.we PEGylated the only free thiol group at Cysteine121 of bovine milk β-Lg in a mono-site-specific manner using methoxy PEG-Maleimide(mPEG-MAL)with two different molecular weights(Mw,5,000 and 10,000).First optimal modification condition were optimized by doing single factor optimization experiment,then the resulting products were purified by cation exchange chromatography and verified by SDS-PAGE analysis and MALDI-TOF mass spectrometry.The antigenicity of PEGylated protein was analyzed by indirect competitive ELISA.To explain the alteration in antigenicity and the antigenic response mechanism of PEGylated protein in depth,the conformational changes were characterized.Conformational changes were investigated by circular dichroism(CD)spectroscopy,free SH group analysis,intrinsic fluorescence analysis,and surface hydrophobicity analysis.The relationship among the conjugation site,conformational changes,and antigenicity of cysteine-specific PEGylated β-Lg was discussed and illustrated by theoretical derivation.We hope to provide some theoretical basis for the effective regulation of β-Lg antigenicity by systematically performing site-specific PEGylation at different sites.The main research methods,results and discussions are as follows:1.PEGylated the only free thiol group at Cysteine121 of bovine milk β-LG in a mono-site-specific manner using methoxy PEG-Maleimide(mPEG-MAL)and optimized the reaction conditions to obtain the optimal modification conditions with high modification rate.The apparent relative molecular weight and modification rate of the modified products were characterized bySDS-PAGE analysis and gel filtration chromatography.The modification conditions such as reaction time,reaction pH value,amount of modifier and amount of reducing agent were optimized.The optimization results showed that the amount of modifier and reaction time were the main factors influencing the modification rate.The optimal modification conditions were:β-LG and mPEG-MAL(molar ratio,1:30)were dissolved in the phosphate buffer(0.01 M,pH 7.0)with 3-fold molar of TCEP and 2 mM EDTA.The mixture was incubated at 4℃for 24 h.2.Cation exchange chromatography was used to separate and purify the modified products of 5/10 kDa mPEG-MAL and β-LG,and the purified products were identified by SDS-PAGE combined with MALDI-TOF-MS analysis.SDS-PAGE analysis showed that the modified products of 5 kDa mPEG-MAL and β-LG(β-LG-PEG5K)and 10 kDa mPEG-MAL and β-LG(β-LG-PEG10K)had only one single band,with apparent molecular weights of 28 kDa and 38 kDa,respectively.MALDI-TOF-MS analysis showed that the actual molecular weights of β-LG-PEG5K and β-LG-PEG10K were 23.6kda and 28.7kda.The results showed that all the modified products were single modified products connected with a single PEG molecule and a single protein.The purity of the modified products was over 90%.After modification with mPEG-MAL,the content of the free sulfhydryl group in β-LG-PEG5K and β-LG-PEG10K decreased from 52.34±0.13 mol/g pro to 0.28±0.19 mol/g pro and 0.12±0.08 mol/g pro,indicating that PEG molecules were bound to the free thiol group at Cys121 of β-LG.3.The changes of protein structure of β-LG samples were investigated by circular dichroism spectrum,intrinsic fluorescence spectrum and surface hydrophobicity.The results showed that the protein structure partially unfolded after cysteine-specific PEGylation,the β-strends content decreased about 7%,the unordered content increased by about 6%,and the intrinsic fluorescence intensity was enhanced.Indirect competitive ELISA result showed that the antigenicity of PEGylated β-LG was significantly increased.The relationship between the structural and antigenicity changes were analysised by the experimental results combining theoretical derivation.The results indicated that during the cysteine-specific PEGylation at thiol group of cysteine 121,the unfolded of β-LG resulted in the forming of conformational epitopes thus made the antigenicity increased.At the same time,compared with common covalent modification,site-specific PEGylation can effectively investigate the relationship bewtween differernt binding sites and antigenicity.