Immunochemical Characterization of Two <i>Pichia pastoris-</i>Derived Recombinant Group 5 <i>Dactylis glomerata</i> Isoallergens

Abstract

Background: Grass pollen of the Poaceae grasses are known to be highly allergenic. Major allergens from the species Lolium, Phleum, Poa and Holcus have been cloned and expressed as recombinant proteins, but of the important species Dactylis glomerata no recombinants are available. Methods: Dac g 5 was cloned by PCR on the basis of homology with Lol p 5 and expressed in Pichia pastoris. Recombinant Dac g 5 (rDac g 5) was affinity purified and compared to natural Dac g 5 (nDac g 5) by immunoblot, radioallergosorbent test (RAST), RAST inhibition, basophil histamine release assay (HRA), competitive radioimmunoassay (RIA) and sandwich enzyme-linked immunosorbent assay (ELISA). In addition, N-terminal sequencing, concanavalin A (Con A) binding, circular dichroism spectrum measurements and matrix-assisted laser desorption ionization-time of flight mass-spectrometric analysis were performed. Results: Clones were obtained that coded for pro-Dac g 5 and two mature isoforms of Dac g 5; the deduced amino acid sequences of both isoforms differed by 4 amino acids. Both mature isoforms were expressed in Pichia at a concentration of ∼15 mg/l. SDS-PAGE analysis showed that rDac g 5 had an apparent M_r approximately 10 kD above nDac g 5. By mass spectrometry this difference was shown to be around 2.5 kD. Positive Con A staining suggested (O-linked) glycosylation as an explanation for this increase in M_r. Whereas both purified recombinants showed a tendency to dimerize, purified nDac g 5 contained a 12-kD peptide not observed for rDac g 5. RAST, RAST inhibition and HRA showed that the IgE reactivity of rDac g 5 was similar to that of nDac g 5. A small subgroup, however, clearly demonstrated decreased IgE reactivity to rDac g 5.02. Differences in immune reactivity of both isoforms were confirmed by monoclonal antibody (mAb)-based sandwich ELISA. Conclusions: Dac g 5 was successfully cloned and expressed in P. pastoris. Minor differences in primary structure between isoforms influence their immune reactivity.

The Major Grass Pollen Group 5 Allergen from <i>Dactylis glomerata </i>and Its C-Terminal Split Product Both Behave as Dimers: Implications for Allergen Standardization

“…Elution of group-5-enriched fractions was achieved with water, and was followed by dialysis against PBS. Recombinant Dac g 5.01 was affinity-purified from Pichia pastoris culture supernatant as described elsewhere [14]. …”

“…The 125 I-labeled nDac g 5 gave an elution pattern of three distinct peaks with decreasing M r . The third peak (C-terminal peptide) was used in competitive RIA [14]. …”

“…To date, grass pollen allergens are divided into 11 groups [1, 2, 3, 4, 5], of which group 1 and group 5 have been identified as the most important. Several group 5 molecules have already been purified and/or cloned [6, 7, 8, 9, 10, 11, 12, 13, 14]and are described as a group of nonglycosylated, highly homologous proteins with an apparent molecular mass (M r ) on SDS-PAGE of 25–33 kDa [15, 16, 17]. By immunoblot analysis, group 5 allergens were shown to migrate as a doublet around this M r , and both bands showed IgE-binding capacities [4, 5, 18].…”

“…Other studies, however, suggested the existence of N-terminally truncated group 5 allergens [16, 20]. Another characteristic of group 5 allergens was revealed by immunoblot analysis where group 5 activity was also found at approximately double its M r (∼49 kDa) and half its M r (∼12 kDa) [5, 14, 21, 22]. The nature of these group-5-related structures has not yet been completely elucidated.…”

The Disulphide Mapping, Folding and Characterisation of Recombinant Ber e 1, an Allergenic Protein, and SFA8, Two Sulphur-rich 2S Plant Albumins

“…31,32 In particular, short O-linked saccharides of mannose containing alpha 1,2 glycosidic or phosphorylated mannose linkages have been characterised. 31,33 The published results indicate that the short glycans added by P. pastoris did not alter the 3D structures 31 or the immune reactivity 34,35 of expressed proteins but did reduce the authenticity of product. 36 …”