title: 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.
  
  
  METHODS
  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.affiliation: Department of Biology, Baylor University, Waco, TX 76798, USA.
authors.forename: Zun
authors.initials: Z
authors.lastname: Liu
authors.forename: Shikha
authors.initials: S
authors.lastname: Bhattacharyya
authors.forename: Bo
authors.initials: B
authors.lastname: Ning
authors.forename: Terumi
authors.initials: T
authors.lastname: Midoro-Horiuti
authors.forename: Edmund W
authors.initials: EW
authors.lastname: Czerwinski
authors.forename: Randall M
authors.initials: RM
authors.lastname: Goldblum
authors.forename: Andrew
authors.initials: A
authors.lastname: Mort
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Kearney
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D000485:Allergens / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D052179:Antigens, Plant; D001491:Basophils / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D015550:Cell Degranulation; D002460:Cell Line; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism; D018653:Juniperus / Q000276:immunology; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011133:Polysaccharide-Lyases / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics; D006255:Rhinitis, Allergic, Seasonal / Q000276:immunology*; D014026:Nicotiana; D014027:Tobacco Mosaic Virus / Q000235:genetics*
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D010940:Plant Proteins; D011994:Recombinant Proteins; C121951:jun a 1 protein, Juniperus ashei; D007073:Immunoglobulin E; D011133:Polysaccharide-Lyases; C023911:pectate lyase