Cloning, expression and patient IgE reactivity of recombinant Pru du 6, an 11S globulin from almond

LeAnna N. Willison, Pallavi Tripathi, Girdhari Sharma, Suzanne S Teuber, Shridhar K. Sathe, Kenneth H. Roux

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Background: IgE-reactive proteins have been identified in almond; however, few have been cloned and tested for specific patient IgE reactivity. Here, we clone and express prunin 1 and prunin 2, isoforms of the major almond protein prunin, an 11S globulin, and assay each for IgE reactivity. Methods: Prunin isoforms were PCR-amplified from an almond cDNA library, sequenced, cloned and expressed in Escherichia coli. Reactivity to the recombinant (r) allergens, Pru du 6.01 and Pru du 6.02, was screened by dot blot and immunoblot assays using sera from almond-allergic patients and murine monoclonal antibodies (mAbs). Sequential IgE-binding epitopes were identified by solid-phase overlapping peptide analysis. Epitope stability was assessed by assaying denatured recombinant proteins by immunoblot. Results: IgE reactivity to rPru du 6.01 and rPru du 6.02 was found in 9 of 18 (50%) and 5 of 18 patients (28%), respectively. Four patients (22%) demonstrated reactivity to both isoforms. Murine anti-almond IgG mAbs also showed greater reactivity to rPru du 6.01 than to rPru du 6.02. Both stable and labile epitopes were detected. Six IgE-binding sequential epitope-bearing peptide segments on Pru du 6.01 and 8 on Pru du 6.02 were detected using pooled almond-allergic sera. Conclusions: rPru du 6.01 is more widely recognized than rPru du 6.02 in our patient population. The identification of multiple sequential epitopes and the observation that treatment with denaturing agents had little effect on IgE-binding intensity in some patients suggests an important role for sequential epitopes on prunins.

Original languageEnglish (US)
Pages (from-to)267-281
Number of pages15
JournalInternational Archives of Allergy and Immunology
Volume156
Issue number3
DOIs
StatePublished - Oct 2011

Fingerprint

Globulins
Immunoglobulin E
Organism Cloning
Epitopes
Protein Isoforms
Monoclonal Antibodies
Peptides
Prunus dulcis
Serum
Gene Library
Recombinant Proteins
Allergens
Proteins
Clone Cells
Escherichia coli
Polymerase Chain Reaction
prunin
Population

Keywords

  • 11S globulin
  • Almond
  • Amandin
  • Food allergy
  • IgE
  • Linear epitope
  • Recombinant allergen
  • Solid-phase overlapping peptide analysis
  • Tree nut

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Cite this

Cloning, expression and patient IgE reactivity of recombinant Pru du 6, an 11S globulin from almond. / Willison, LeAnna N.; Tripathi, Pallavi; Sharma, Girdhari; Teuber, Suzanne S; Sathe, Shridhar K.; Roux, Kenneth H.

In: International Archives of Allergy and Immunology, Vol. 156, No. 3, 10.2011, p. 267-281.

Research output: Contribution to journalArticle

Willison, LeAnna N. ; Tripathi, Pallavi ; Sharma, Girdhari ; Teuber, Suzanne S ; Sathe, Shridhar K. ; Roux, Kenneth H. / Cloning, expression and patient IgE reactivity of recombinant Pru du 6, an 11S globulin from almond. In: International Archives of Allergy and Immunology. 2011 ; Vol. 156, No. 3. pp. 267-281.
@article{4c48c61e6e0e41e597677ba3d7e941d2,
title = "Cloning, expression and patient IgE reactivity of recombinant Pru du 6, an 11S globulin from almond",
abstract = "Background: IgE-reactive proteins have been identified in almond; however, few have been cloned and tested for specific patient IgE reactivity. Here, we clone and express prunin 1 and prunin 2, isoforms of the major almond protein prunin, an 11S globulin, and assay each for IgE reactivity. Methods: Prunin isoforms were PCR-amplified from an almond cDNA library, sequenced, cloned and expressed in Escherichia coli. Reactivity to the recombinant (r) allergens, Pru du 6.01 and Pru du 6.02, was screened by dot blot and immunoblot assays using sera from almond-allergic patients and murine monoclonal antibodies (mAbs). Sequential IgE-binding epitopes were identified by solid-phase overlapping peptide analysis. Epitope stability was assessed by assaying denatured recombinant proteins by immunoblot. Results: IgE reactivity to rPru du 6.01 and rPru du 6.02 was found in 9 of 18 (50{\%}) and 5 of 18 patients (28{\%}), respectively. Four patients (22{\%}) demonstrated reactivity to both isoforms. Murine anti-almond IgG mAbs also showed greater reactivity to rPru du 6.01 than to rPru du 6.02. Both stable and labile epitopes were detected. Six IgE-binding sequential epitope-bearing peptide segments on Pru du 6.01 and 8 on Pru du 6.02 were detected using pooled almond-allergic sera. Conclusions: rPru du 6.01 is more widely recognized than rPru du 6.02 in our patient population. The identification of multiple sequential epitopes and the observation that treatment with denaturing agents had little effect on IgE-binding intensity in some patients suggests an important role for sequential epitopes on prunins.",
keywords = "11S globulin, Almond, Amandin, Food allergy, IgE, Linear epitope, Recombinant allergen, Solid-phase overlapping peptide analysis, Tree nut",
author = "Willison, {LeAnna N.} and Pallavi Tripathi and Girdhari Sharma and Teuber, {Suzanne S} and Sathe, {Shridhar K.} and Roux, {Kenneth H.}",
year = "2011",
month = "10",
doi = "10.1159/000323887",
language = "English (US)",
volume = "156",
pages = "267--281",
journal = "International Archives of Allergy and Immunology",
issn = "1018-2438",
publisher = "S. Karger AG",
number = "3",

}

TY - JOUR

T1 - Cloning, expression and patient IgE reactivity of recombinant Pru du 6, an 11S globulin from almond

AU - Willison, LeAnna N.

AU - Tripathi, Pallavi

AU - Sharma, Girdhari

AU - Teuber, Suzanne S

AU - Sathe, Shridhar K.

AU - Roux, Kenneth H.

PY - 2011/10

Y1 - 2011/10

N2 - Background: IgE-reactive proteins have been identified in almond; however, few have been cloned and tested for specific patient IgE reactivity. Here, we clone and express prunin 1 and prunin 2, isoforms of the major almond protein prunin, an 11S globulin, and assay each for IgE reactivity. Methods: Prunin isoforms were PCR-amplified from an almond cDNA library, sequenced, cloned and expressed in Escherichia coli. Reactivity to the recombinant (r) allergens, Pru du 6.01 and Pru du 6.02, was screened by dot blot and immunoblot assays using sera from almond-allergic patients and murine monoclonal antibodies (mAbs). Sequential IgE-binding epitopes were identified by solid-phase overlapping peptide analysis. Epitope stability was assessed by assaying denatured recombinant proteins by immunoblot. Results: IgE reactivity to rPru du 6.01 and rPru du 6.02 was found in 9 of 18 (50%) and 5 of 18 patients (28%), respectively. Four patients (22%) demonstrated reactivity to both isoforms. Murine anti-almond IgG mAbs also showed greater reactivity to rPru du 6.01 than to rPru du 6.02. Both stable and labile epitopes were detected. Six IgE-binding sequential epitope-bearing peptide segments on Pru du 6.01 and 8 on Pru du 6.02 were detected using pooled almond-allergic sera. Conclusions: rPru du 6.01 is more widely recognized than rPru du 6.02 in our patient population. The identification of multiple sequential epitopes and the observation that treatment with denaturing agents had little effect on IgE-binding intensity in some patients suggests an important role for sequential epitopes on prunins.

AB - Background: IgE-reactive proteins have been identified in almond; however, few have been cloned and tested for specific patient IgE reactivity. Here, we clone and express prunin 1 and prunin 2, isoforms of the major almond protein prunin, an 11S globulin, and assay each for IgE reactivity. Methods: Prunin isoforms were PCR-amplified from an almond cDNA library, sequenced, cloned and expressed in Escherichia coli. Reactivity to the recombinant (r) allergens, Pru du 6.01 and Pru du 6.02, was screened by dot blot and immunoblot assays using sera from almond-allergic patients and murine monoclonal antibodies (mAbs). Sequential IgE-binding epitopes were identified by solid-phase overlapping peptide analysis. Epitope stability was assessed by assaying denatured recombinant proteins by immunoblot. Results: IgE reactivity to rPru du 6.01 and rPru du 6.02 was found in 9 of 18 (50%) and 5 of 18 patients (28%), respectively. Four patients (22%) demonstrated reactivity to both isoforms. Murine anti-almond IgG mAbs also showed greater reactivity to rPru du 6.01 than to rPru du 6.02. Both stable and labile epitopes were detected. Six IgE-binding sequential epitope-bearing peptide segments on Pru du 6.01 and 8 on Pru du 6.02 were detected using pooled almond-allergic sera. Conclusions: rPru du 6.01 is more widely recognized than rPru du 6.02 in our patient population. The identification of multiple sequential epitopes and the observation that treatment with denaturing agents had little effect on IgE-binding intensity in some patients suggests an important role for sequential epitopes on prunins.

KW - 11S globulin

KW - Almond

KW - Amandin

KW - Food allergy

KW - IgE

KW - Linear epitope

KW - Recombinant allergen

KW - Solid-phase overlapping peptide analysis

KW - Tree nut

UR - http://www.scopus.com/inward/record.url?scp=79959634223&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=79959634223&partnerID=8YFLogxK

U2 - 10.1159/000323887

DO - 10.1159/000323887

M3 - Article

C2 - 21720172

AN - SCOPUS:79959634223

VL - 156

SP - 267

EP - 281

JO - International Archives of Allergy and Immunology

JF - International Archives of Allergy and Immunology

SN - 1018-2438

IS - 3

ER -