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 Citations (Scopus)

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

Fingerprint

Allergens
Immunoglobulin E
Serum
Juniperus
Tobacco Mosaic Virus
Agrobacterium tumefaciens
Musa
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Extracellular Fluid
Syringes
Capillary Electrophoresis
Therapeutic Uses
Pollen
Recombinant Proteins
Tobacco
Polyacrylamide Gel Electrophoresis
Mass Spectrometry
Necrosis
Complementary DNA
Bacteria

Keywords

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

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Cite this

Plant-expressed recombinant mountain cedar allergen jun a 1 is allergenic and has limited pectate lyase activity. / Liu, Zun; Bhattacharyya, Shikha; Ning, Bo; Midoro-Horiuti, Terumi; Czerwinski, Edmund W.; Goldblum, Randall M.; Mort, Andrew; Kearney, Christopher M.

In: International Archives of Allergy and Immunology, Vol. 153, No. 4, 11.2010, p. 347-358.

Research output: Contribution to journalArticle

Liu, Zun ; Bhattacharyya, Shikha ; Ning, Bo ; Midoro-Horiuti, Terumi ; Czerwinski, Edmund W. ; Goldblum, Randall M. ; Mort, Andrew ; Kearney, Christopher M. / Plant-expressed recombinant mountain cedar allergen jun a 1 is allergenic and has limited pectate lyase activity. In: International Archives of Allergy and Immunology. 2010 ; Vol. 153, No. 4. pp. 347-358.
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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.",
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T1 - Plant-expressed recombinant mountain cedar allergen jun a 1 is allergenic and has limited pectate lyase activity

AU - Liu, Zun

AU - Bhattacharyya, Shikha

AU - Ning, Bo

AU - Midoro-Horiuti, Terumi

AU - Czerwinski, Edmund W.

AU - Goldblum, Randall M.

AU - Mort, Andrew

AU - Kearney, Christopher M.

PY - 2010/11

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N2 - 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.

AB - 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.

KW - Agrobacterium

KW - Apoplast

KW - Immunoglobulin E

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KW - Juniperus ashei

KW - Necrosis

KW - Nicotiana benthamiana

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