Isoform identification and characterization of Art v 3, the lipid-transfer protein of mugwort pollen.

Art v 3, the lipid-transfer protein (LTP) of Artemisia vulgaris pollen is a relevant allergen showing frequent cross-reactivity with homologues in other plants. Here we report the identification of four full-length Art v 3 sequences obtained by cDNA cloning using mass spectrometry-based sequencing. Two isoforms, Art v 3.0201 and Art v 3.0301 were expressed as soluble proteins in Escherichia coli Rosetta-gami B(DE3) pLysS using different expression systems. Purified natural and recombinant Art v 3 demonstrated similar secondary structures in circular dichroism analysis. All preparations showed high thermal stability but low resistance to gastric digestion with pepsin. Patient-specific IgE reactivity patterns to natural or recombinant isoallergens were observed among Art v 3-sensitized subjects. Using Immuno Solid-phase Allergen Chip (ISAC) assays, frequent cross-reactivity of Art v 3 with LTPs from peach and hazelnut was shown. The biological activity of both isoforms was comparable to the natural allergen in basophil release assays. The newly identified sequences provide the basis for recombinant mugwort LTP production enabling batch-to-batch reproducibility and thus ensuring high-quality products for diagnosis and therapy.

Influence of the natural ripening stage, cold storage, and ethylene treatment on the protein and IgE-binding profiles of green and gold kiwi fruit extracts.

Kiwi fruit is an important source of food allergens, the number and relevance of which are still the object of investigation. Following a comparative analysis of the protein profiles in SDS-PAGE and IgE immunoblotting, a significant influence of conditions such as the ripening stage and the extraction method on the composition of green and gold kiwi fruit extracts was observed. Furthermore, the experimental data indicate that, mostly in the green species, a ripe fruit may have a different concentration of total proteins and a different amount of single components when ripeness is reached by different means of postharvest handling, such as ethylene exposure with or without previous cold storage. In summary, this study emphasizes the level of complexity associated with the preparation of extracts when a known and defined concentration of proteins/allergens is requested.

Kiwellin, a modular protein from green and gold kiwi fruits: evidence of in vivo and in vitro processing and IgE binding.

Kiwellin, an allergenic protein formerly isolated from green kiwi fruit, has been identified as the most abundant component of the gold kiwi species. A protein named KiTH, showing a 20 kDa band on reducing SDS-PAGE and 100% identity with the C-terminal region of kiwellin, has been identified in the extract of the ripe green species. In vitro treatment of purified kiwellin with the protease actinidin from green kiwi fruit originated KiTH and kissper, a recently described pore-forming peptide. Primary structure analysis and experimental evidence suggest that kiwellin is a modular protein with two domains. It may undergo in vivo proteolytic processing by actinidin, thus producing KiTH and kissper. When probed with sera recognizing kiwellin from green kiwi fruit, KiTH showed IgE binding, with reactivity levels sometimes different from those of kiwellin. The IgE-binding capacity of kiwellin from gold kiwi fruit appears to be similar to that of the green species.