The Effect Of PEG Modification On Antigenicity And Conformation Changes Of β-Lactoglobulin

PEGylation is a new technique which developed in the late1970s and can improve the pharmaceutical proteins by reducing or removing antigenicity, increasing water solubility, and improving stability. In this paper, the influence of PEGylation on the antigenicity and changes in conformation of β-lactoglobulin was studied. The relationship between the antigenicity of β-lactoglobulin and changes in conformation were discussed preliminary.β-lactoglobulin is the main allergen in milk, so eliminating the allergenicity of β-lactoglobulin has always been one of the hot topics in research of international dairy producing technology. In this paper,β-lactoglobulin was modified by mPEG-SC(20kDa) and changes of antigenicity was studied by indirect competitive ELISA. The results indicated that the antigenicity was affected the most by time. The optimal modification condition was pH7.0, reacting time8h, the mass ratio of mPEG-SC and β-Lg3:1with the antigenicity of53.64μg/mL (initial value180μg/mL). The reduction rate of antigenicity was70.20%. There were two products in SDS-PAGE of β-Lg modified by PEG:di-PEGylated β-Lg(58kDa) and tri-PEGylated β-Lg(78kDa).The product of β-Lg modified by PEG was separated by cationic exchange chromatography (SP Sepharose Fast Flow). The elution peaks were collected. SDS-PAGE had confirmed that two-point modified product and three-point modified product were obtained. Fluorescence spectrum experiment of modified product indicated fluorescence quenching occurred during PEGylation which led to the changes in tertiary structure and quaternary structure of β-Lg. In the meanwhile, the relative fluorescence intensity decreased with the decreasing of the antigenicity.The results of circular dichroism spectrum indicated, during PEGylation, from β-Lg to di-PEGylated β-Lg, to tri-PEGylated β-Lg, the contents of β-sheet increased gradually (β-sheet contents:β-Lg33.2%, di-PEGylated β-Lg35.7%, tri-PEGylated β-Lg44.6%). With the increasing of PEG on β-Lg, the contents of a-helix changed from14.3%to14.9%, to19.2%indicating gradually increasing ofa-helix. In a word, the research on conformation indicated that comformational epitopes of β-Lg changed after PEG modification which led to the decreasing of the antigenicity, that was antigenic determinants were covered after PEG modification which decreased the immunity and antigenicity of β-Lg.