Plant-expressed recombinant mountain cedar allergen jun a 1 is allergenic and has limited pectate lyase activity

Zun Liu, Shikha Bhattacharyya, Bo Ning, Terumi Midoro-Horiuti, Edmund W. Czerwinski, Randall M. Goldblum, Andrew Mort, Christopher M. Kearney

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

Background: Mountain cedar (Juniperus ashei) pollen commonly causes a winter time allergic rhinitis in the central USA. Jun a 1 is the dominant allergenic protein, but biologically active recombinant Jun a 1 has not been successfully expressed, despite numerous attempts with several expression systems. Method: Jun a 1 cDNA was inserted into a tobacco mosaic virus vector and transferred to Agrobacterium tumefaciens. Bacteria were syringe-inoculated into leaves of Nicotiana benthamiana (agroinoculation). The interstitial (apoplastic) fluid containing Jun a 1 was isolated. The recombinant protein was analyzed by SDS-PAGE, N-terminal sequencing and MALDI-TOF to confirm identity. Immunogenicity was examined with IgE from allergic patient's sera, mouse monoclonal anti-Jun a 1 antibodies, IgE-binding inhibition and by degranulation of RBL SX-38 cells sensitized with sera from allergic patients. Pectate lyase activity was assayed by capillary zone electrophoresis and mass spectrometry analysis. Results: Recombinant Jun a 1 was recovered in good quantity (100 μg/g leaf material), was confirmed as Jun a 1, bound IgE from sera from cedar hypersensitive patients and inhibited IgE binding to native Jun a 1. Jun a 1 mutants were created and their pectate lyase activity quantified. For the first time, Jun a 1 pectate lyase activity was demonstrated, which may explain the necrosis seen on host plants, which was similar to that of control plants expressing banana pectate lyase. Conclusions: A means of producing recombinant Jun a 1 is now available for structure/function studies and potentially for diagnostic and therapeutic uses.

Original languageEnglish (US)
Pages (from-to)347-358
Number of pages12
JournalInternational Archives of Allergy and Immunology
Volume153
Issue number4
DOIs
StatePublished - Nov 2010

Keywords

  • Agrobacterium
  • Apoplast
  • Immunoglobulin E
  • Jun a 1
  • Juniperus ashei
  • Necrosis
  • Nicotiana benthamiana

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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