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

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.

authors

Liu Z,Bhattacharyya S,Ning B,Midoro-Horiuti T,Czerwinski EW,Goldblum RM,Mort A,Kearney CM

doi

10.1159/000316345

subject

Has Abstract

pub_date

2010-01-01 00:00:00

pages

347-58

issue

4

eissn

1018-2438

issn

1423-0097

pii

000316345

journal_volume

153

pub_type

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