The lipid transfer protein of apple fruit, Mal d 3, has been produced as a soluble recombinant protein in transformed Escherichia coli cells using the GATEWAY (TM) technology. Circular dichroism spectra showing the protein essentially consists of alpha-helices indicate that the rMal d 3 is properly folded. The H-1 NMR spectra also indicates a correct fold for the recombinant allergen. The reactivity of rMal d 3 towards IgE from apple allergic patients and in vitro degranulation activity measured on transformed rat basophil leukemia cells expressing the human Fc epsilon RI alpha-subunit of rMal d 3 is similar to those of the native allergen purified from apple fruits. The expression of active rMal d 3 in E. coli is readily feasible and offers an interesting alternative to the production of recombinant allergen in the yeast Pichia pastoris. This expression in E coli open the way to the modification of Mal d 3 by site-directed mutagenesis for immunotherapy purposes. (C) 2009 Elsevier Inc. All rights reserved.