Immunological Comparison of Native and Recombinant Hen's Egg Yolk Allergen, Chicken Serum Albumin (Gal d 5), Produced in Kluveromyces lactis

Abstract

Chicken serum albumin (CSA) is a hen's egg yolk allergen causing IgE-mediated allergy. The objective of this study was to produce a recombinant version of CSA and compare its IgE reactivity to natural CSA (nCSA). CSA was cloned and expressed as a soluble fraction in the yeast Kluyveromyces lactis (K. lactis) protein expression system. The gene encoding CSA was amplified with a C-terminal hemagglutinin epitope tag by polymerase chain reaction (PCR) and cloned into the pKLAC2 expression vector prior to transforming into K. lactis. Recombinant CSA (rCSA) was purified by immunoprecipitation. Twenty-one patients allergic to hen's egg white were examined for sensitisation against nCSA. 38% of patients were found to be sensitised to CSA based on Western immunoassay. Immunoglobulin E (IgE) binding capacity of rCSA and nCSA was analysed by ELISA using sera from patients sensitised to CSA. Levels of IgE-binding were similar for both the recombinant and the natural CSA, indicating the existence of similar epitopes. rCSA produced in this study is a potential candidate to be used in component-resolved diagnosis (CRD) of egg yolk allergy. The usefulness of rCSA in CRD of egg yolk allergy warrants further characterisation using sera from patients with allergy to hen's egg yolk in future studies.

Keywords: allergy; chicken serum albumin; egg yolk allergens; recombinant allergens.

Figure 1

Time course of expression of K. lactis culture supernatant fractions containing secreted rCSA by (A) SDS-PAGE and (B) Western immunoblot analysis. Lane 1–6: galactose-induced culture supernatants from day 2–7. (C) SDS-PAGE analysis of purified nCSA. The arrows indicate the positions of both rCSA and nCSA at 69 kDa. MW: molecular weight marker SeeBlue^® Pre-Stained Protein Standard. MW: molecular weight marker; rCSA: recombinant chicken serum albumin; SDS-PAGE: sodium dodecyl sulphate gel electrophoresis; nCSA: natural chicken serum albumin.

Figure 2

Immunoprecipitation analysis of purified rCSA fractions by (A) SDS-PAGE and (B) Western immunoblotting. Lane 1: unbound sample, Lane 2: elution containing rCSA. Arrows indicate the rCSA. MW: molecular weight marker SeeBlue^® Pre-Stained Protein Standard.

Figure 3

(A) IgE reactivity to nCSA in patients sensitised to hen’s egg white determined by dot blot. The grid above shows the patient ID described in Table 1 corresponding to each spot. nCSA-sensitised patients are highlighted in the grid. Dots denoted by N are sera from nonallergic (control) subjects. A to C represent row identity and I to VIII represent column identity in the grid and the corresponding Western dot blot; (B) IgE reactivity to rCSA of sera from patients sensitised to nCSA described in Figure 3 by Western dot-blot analysis. Dots denoted by N are sera from nonallergic healthy (control) subjects.

Figure 3

(A) IgE reactivity to nCSA in patients sensitised to hen’s egg white determined by dot blot. The grid above shows the patient ID described in Table 1 corresponding to each spot. nCSA-sensitised patients are highlighted in the grid. Dots denoted by N are sera from nonallergic (control) subjects. A to C represent row identity and I to VIII represent column identity in the grid and the corresponding Western dot blot; (B) IgE reactivity to rCSA of sera from patients sensitised to nCSA described in Figure 3 by Western dot-blot analysis. Dots denoted by N are sera from nonallergic healthy (control) subjects.

Figure 4

Comparison of the binding activity of human sIgE against recombinant CSA and natural CSA. N denotes serum from healthy subjects and numbers denote different patients’ sera sensitised to nCSA. Data are average of triplicate mean +/− SEM. sIgE: specific immunoglobulin E; SEM: standard error of mean.