Effect Of Site Specific PEGylation Of β-lactoglobulin At Glutamine Residues On Its Antigenicity

Bovineβ-lactoglobulin（β-LG）is one of the main allergens in milk.Using covalent modification to reduce the antigenicity ofβ-LG has been a hotspot of international dairy products processing researches.In this study,enzymatic PEGylation was applied to modifyβ-LG in order to reduce its antigenicity.5 kDa methoxy polyethylene glycol-amine（mPEG-NH₂）was used to modifyβ-LG at glutamine residues（Gln）under the catalysis of transglutaminase（TGase）.After PEGylation,the mono-PEGylatedβ-LG was obtained.Then,the structural characterization,PEGylation site and antigenicity analysis were performed on the obtained PEGylatedβ-LG.The relationship between PEGylation site,structural changes and antigenicity ofβ-LG during enzymatic PEGylation were investigated and the mechanism of antigenicity changes ofβ-LG during PEGylation was explored.Firstly,β-LG was site specifically PEGylated at Gln and the PEGylation conditions were optimized.Taking the modification rate of PEGylatedβ-LG as an index,the modification conditions were optimized by SDS-PAGE and gel filtration chromatography analysis.The results showed that the molar ratio ofβ-LG to PEG,the mass ratio of enzyme to substrate,the reaction temperature,pH and the addition amount of ethanol all had great influence on the modification reaction.The optimal modification conditions were obtained as follows:reaction pH was 7.0,reaction temperature was 25℃,molar ratio ofβ-LG to PEG was 1:15,mass ratio of TGase toβ-LG was 3:1,the amount of ethanol added in buffer was 25%.Under the optimal modification conditions,the modification rate of PEGylatedβ-LG can reach to60.17%and only one kind of PEGylatedβ-LG was formed.The PEGylatedβ-LG was purified by cation exchange chromatography and the obtained PEGylatedβ-LG was identified.The SDS-PAGE analysis result indicated that the purified PEGylated product showed only one single band,and its apparent molecular mass was about 28 kDa.The MALDI-TOF-MS result showed that the actual molecular mass of nativeβ-LG and the PEGylatedβ-LG were about 18.3 kDa and 23.4 kDa,respectively,which indicated that the modified product was mono-PEGylatedβ-LG with only one PEG conjugated.The result of RP-HPLC showed that the obtained mono-PEGylatedβ-LG is homogeneous and has no other position isomers.The relationship between PEGylation site,structural changes and antigenicity ofβ-LG during enzymatic PEGylation were investigated by circular dichroism,intrinsic fluorescence spectroscopy,surface hydrophobicity,free sulfhydryl content measurement,PEGylation site analysis and antigenicity measurement.Structure analysis showed that the secondary structure ofβ-LG didn’t change obviously after PEGylation,while the intrinsic fluorescence intensity decreased,the surface hydrophobicity increased,and the free sulfhydryl content didn’t change significantly,which indicated that the tertiary and quarternary structures ofβ-LG slightly changed after PEGylation.The PEGylation site analysis showed that the Gln155 or Gln159might be the most possible PEGylation site.The antigenicity analysis showed that the antigenicity ofβ-LG was significantly decreased by 69%after enzymatic PEGylation.Combining the results of structure and antigenicity analysis ofβ-LG before and after PEGylation,it can be concluded that the antigenicity decrease mechanism ofβ-LG induced by TGase-catalyzed PEGylation was mainly due to the steric shielding effect of PEG chains.The long PEG chains with large molecular mass conjugated toβ-LG at glutamine residues covered on the surface of the protein and masked part of linear epitopes and conformational epitopes on the surface ofβ-LG,which hindered their binding to specific antibodies thus leading to the reduction of its antigenicity.