Isolation and biochemical characterization of a thaumatin-like kiwi allergen

Background: Kiwi fruit allergy, as well as its association with hypersensitivity to other foods and to pollen, has been extensively reported in the last few years. Several IgE-binding components have been detected in kiwi extract, but only one 30-kd allergen has been isolated; it was identified as actinidin (Act c 1). Recently, we have reported a 24-kd kiwi protein to be a potential major allergen in a group of patients with oral allergy syndrome (OAS). Objective: The aim of this study was to purify and characterize the 24-kd kiwi allergen biochemically. Methods: Seven polysensitized patients with OAS to kiwi were used in this study. The kiwi allergen was isolated by using a combination of gel permeation, ion exchange, and immobilized metal ion affinity chromatography. Its biochemical characterization included determination of its isoelectric point, molecular weight, N-terminal sequencing, concanavalin A-binding ability, digestibility in simulated gastric fluid, and antifungal activity. Western blotting, 2-dimensional PAGE immunoblotting, and skin prick tests were performed to characterize the isolated protein immunochemically. Results: All 7 patients recognized the isolated 24-kd kiwi protein as an allergen. The isolated protein consisted of 2 isoforms with isoelectric points of 9.4 and 9.5 migrated as one protein band of 20 kd after SDS-PAGE under nonreducing conditions or at 24 kd under reducing conditions. The partial N-terminal sequence revealed that it is a thaumatin-like protein (TLP) with concanavalin A-binding ability. The protein showed antifungal activity toward Saccharomyces carlsbergensis, and Candida albicans. The protein was degraded by the simulated gastric fluid within 1 minute. Both isoforms bound IgE from a pool of sera in a 2-dimensional PAGE inummoblot. The TLP elicited positive skin prick test responses in 4 (80%) of 5 patients with OAS. Conclusion: This study reported isolation and full characterization of a new kiwi allergen, TLP (isoelectric points of 9.4 and 9.5 and molecular weight of 24 kd), which belongs to the family of pathogenesis-related proteins. The isolated protein expressed antifungal activity toward S carlsbergensis and C albicans.