Reference "Immunochemical characterization of two Pichia pastoris-derived recombinant group 5 Dactylis glomerata isoallergens"

Reference ID

9603

Title

Immunochemical characterization of two Pichia pastoris-derived recombinant group 5 Dactylis glomerata isoallergens

Source

International archives of allergy and immunology, 2001, vol. 126, pp. 196-205

Authors (3)

Lerouge-P	Faye-L
Aalberse-R-C

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 approximately 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.