Plant-based heterologous expression of Mal d 2, a thaumatin-like protein and allergen of apple (Malus domestica), and its characterization as an antifungal protein

Monika Krebitz, Birgit Wagner, Fatima Ferreira, Clemens Peterbauer, Nuria Campillo, Michael Witty, Daniel Kolarich, Herta Steinkellner, Otto Scheiner, Heimo Breiteneder*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

118 Citations (Scopus)

Abstract

Mal d 2 is a thaumatin-like protein and important allergen of apple fruits that is associated with IgE-mediated symptoms in apple allergic individuals. We obtained a full-length cDNA clone of Mal d 2 from RNA isolated from ripe apple (Malus domestica cv. Golden Delicious). The cDNA's open reading frame encodes a protein of 246 amino acid residues including a signal peptide of 24 residues and two putative glycosylation sites. The deduced amino acid sequence of the mature Mal d 2 protein results in a predicted molecular mass of 23,210.9Da and a calculated pI of 4.55. Sequence comparisons and molecular modeling place Mal d 2 among those pathogenesis-related thaumatin-like proteins that contain a conserved acidic cleft. In order to ensure the correct formation of the protein's eight conserved disulfide bridges we expressed Mal d 2 in Nicotiana benthamiana plants by the use of a tobacco mosaic viral vector. Transfected N.benthamiana plants accumulated Mal d 2 to levels of at least 2% of total soluble protein. MALDI-TOF mass spectrometric analyses of the recombinant Mal d 2 and its proteolytic fragments showed that the apple-specific leader peptide was correctly cleaved off by the host plant and that the mature recombinant protein was intact and not glycosylated. Purified recombinant Mal d 2 displayed the ability to bind IgE from apple-allergic individuals equivalent to natural Mal d 2. In addition, the recombinant thaumatin-like Mal d 2 exhibited antifungal activity against Fusarium oxysporum and Penicillium expansum, implying a function in plant defense against fungal pathogens.

Original languageEnglish
Pages (from-to)721-730
Number of pages10
JournalJournal of molecular biology
Volume329
Issue number4
DOIs
Publication statusPublished - 13 Jun 2003

Keywords

  • Antifungal pathogenesis-related protein
  • Recombinant apple allergen
  • Structural modeling
  • Tobacco mosaic virus
  • Transient expression

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