Plant-expressed recombinant mountain cedar allergen Jun a 1 is allergenic and has limited pectate lyase activity.
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.
Liu
Zun
Z
Department of Biology, Baylor University, Waco, TX 76798, USA.
Bhattacharyya
Shikha
S
Ning
Bo
B
Midoro-Horiuti
Terumi
T
Czerwinski
Edmund W
EW
Goldblum
Randall M
RM
Mort
Andrew
A
Kearney
Christopher M
CM
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*
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