Site specific PEGylation of β-lactoglobulin at glutamine residues and its influence on conformation and antigenicity

beta-lactoglobulin (beta-LG) is one of the main allergens in milk. Polyethylene glycol (PEG) modification (PEGylation) was found to have the ability to reduce the antigenicity of proteins. To determine the effect of site specific PEGylation on beta-LG antigenicity and conformation, we applied 5 kDa methoxy polyethylene glycol-amine (mPEG-NH2) to modify beta-LG at glutamine (Gln) residues under the catalysis of transglutaminase. The antigenicity of beta-LG was measured using rabbit IgG antibodies by indirect competitive ELISA. The result indicated that the antigenicity of beta-LG was decreased from 72.2 mu g/mL to 22.7 mu g/mL after PEGylation. SDS-PAGE and MALDI-TOF-MS showed that the molecular mass of native beta-LG was about 18.3 kDa while the PEGylated beta-LG had a molecular mass of 23.4 kDa, which meant that mono-PEGylated beta-LG was obtained after PEGylation and purification by cation exchange chromatography. Additionally, the circular dichroism spectrum of the PEGylated beta-LG was approximately superimposed on that of beta-LG and the secondary structure content of beta-LG also had no significant changes after PEGylation, which indicated that the secondary structure of beta-LG was preserved. After PEGylation, the intrinsic fluorescence intensity of beta-LG decreased from 6361 to 5159 while the surface hydrophobicity increased, which indicated that the tertiary structure of beta-LG was slightly changed. PEGylation site analysis result showed that Gln 155 or Gln 159 might be the most possible binding site. In conclusion, the decrease of the antigenicity of beta-LG induced by the PEGylation is mainly due to the steric shielding effect of PEG chain rather than conformational changes of beta-LG.