Transgenic Rice Seeds Accumulating Recombinant Hypoallergenic Birch Pollen Allergen Bet v 1 Generate Giant Protein Bodies

A versatile hypoallergenic allergen derivative against multiple allergens is an ideal tolerogen for allergen-specific immunotherapy. Such a tolerogen should exhibit high efficacy, without side effects, when administered at high doses and should be applicable to several allergens. Tree pollen chimera 7 (TPC7), a hypoallergenic Bet v 1 tolerogen against birch pollen allergy, was previously selected by DNA shuffling of 14 types of Fagales tree pollen allergens. In this study, transgenic rice seed accumulating TPC7 was generated as an oral vaccine against birch pollen allergy by expressing this protein as a secretory protein using the N-terminal signal peptide and the C-terminal KDEL tag under the control of an endosperm-specific glutelin promoter. The highest level of TPC7 accumulation was approximately 207 mu g grain(-1). Recombinant TPC7 is a glycoprotein with high mannose-type N-glycan, but without beta 1,2-xylose or alpha 1,3-fucose, suggesting that TPC7 is retained in the endoplasmic reticulum (ER). TPC7 is deposited as a novel, giant spherical ER-derived protein body, > 20 mu m in diameter, which is referred to as the TPC7 body. Removal of the KDEL retention signal or mutation of a cysteine residue resulted in an alteration of TPC7 body morphology, and deletion of the signal peptide prevented the accumulation of TPC7 in rice seeds. Therefore, the novel TPC7 bodies may have formed aggregates within the ER lumen, primarily due to the intrinsic physicochemical properties of the protein.