Digestibility of Natural and Recombinant Allergenic Peanut Proteins in Artificial Gastrointestinal Fluids

Safety assessments are necessary for genetically modified foods in many countries, including Japan. Stabilities during pepsin, trypsin, or pancreatin digestion are a key criterion for assessing the allergenic potential of newly expressed proteins (NEPs). In digestibility tests, NEPs produced by heterologous expression systems were frequently used. Polyhistidine tags (His-tags) are primarily/often used to purify recombinant proteins. Studies of His-tags' influences remain limited on the susceptibility of a protein to pepsin/trypsin digestion, although His-tags can affect protein folding and stability. In this study, we compared the digestibility of the natural peanut allergenic proteins Ara h 1 and Ara h 2 to the recombinant Ara h 1 protein with N-terminal His-tag and recombinant Ara h 2 protein with C-terminal His-tag, respectively. Peptides after the proteolysis were then analyzed using liquid chromatography-tandem mass spectrometry to determine the proteolytic cleavage sites. Differences were detected in the C-terminal region after pepsin cleavage of the His-tag extension of Ara h 1 and Ara h 2 proteins. No differences were observed in other cleavage sites between the natural and recombinant Ara h 1 and Ara h 2 proteins. The N-terminal region of Ara h 1 and Ara h 2, at which the epitopes recognized by most patients allergic to peanut were located, was equally resistant to pepsin digestion regardless of the natural or recombinant forms. In this study, an unintended short protein isoform was detected in the recombinant Ara h 2 samples. This short recombinant isoform may be misfolded, and it showed reduced susceptibility to pepsin digestion relative to natural full-length Ara h 2. In this short Ara h 2 isoform, newly paired disulfide bonds may make it more rigid. Recombinant proteins with His-tags can provide nearly comparable results to the corresponding natural proteins in protease digestions and thus offer information useful for safety assessment.