Recombinant Fusion Proteins Assembling Der p 1 and Der p 2 Allergens from <i>Dermatophagoides pteronyssinus</i>

Abstract

Background: Fusion proteins assembling multiple allergens can be engineered by recombinant DNA technologies in order to produce tools for diagnostic and immunotherapeutic purposes. Herein, we developed and characterized chimeras assembling Der p 1 and Der p 2 allergens as potential candidate vaccines against house dust mite allergy. Methods: Fusion proteins encompassing Der p 2 with either mature or proDer p 1 were expressed in Escherichia coli or Pichia pastoris. Forms with mutation in Der p 1 catalytic site were also engineered. Purified chimeras were characterized by immunoblotting, circular dichroism, disulfide bond mapping, basophil and T lymphocyte stimulation assays. Results: Four fusion proteins were expressed in E. coli as inclusion bodies, whereas only chimeras comprising proDer p 1 were obtained in yeast. All such hybrids formed polymers and aggregates, and yeast-expressed chimeras were unstable. Circular dichroism analysis performed after refolding of bacteria expressed chimeras encompassing mature Der p 1 confirmed partial folding, consistent with the occurrence of both correct and inappropriate intramolecular disulfide bonds. All fusion molecules were recognized by Der p 1- and Der p 2-specific human IgEs, monoclonal and polyclonal antibodies. Fusion proteins activate basophils from mite-allergic patients and trigger the proliferation of specific CD4+ T cells, albeit to a lower level when compared to individual allergens. Conclusions: Production of multiple Der p 1-Der p 2 fusion proteins exhibiting partial folding and proper antigenic properties has been achieved. Nonetheless, significant solubility and stability issues currently limit the application of such chimeras for immunotherapy or diagnostic.

Structural and Functional Characterization of the Major Allergen Amb a 11 from Short Ragweed Pollen

“…As such, we confirm the importance of this molecule in ragweed pollen allergy. done as described previously (10 Choudhury et al (11), pro-rAmb a 11 was purified by immobilized metal affinity chromatography on a HisTrap HP column (GE Healthcare) under denaturing conditions (50 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 20 mM imidazole, 7 M urea) and then eluted with an 0.02-0.5 M imidazole gradient. Eluted fractions containing pro-rAmb a 11 were pooled and incubated with 10 mM DTT at 37°C for 1 h. The protein solution was diluted to a 50 g/ml maximum concentration in a refolding buffer (50 mM Tris-HCl, pH 8.5, 0.5 M NaCl, 5 mM EDTA, 1 mM GSH, 0.1 mM GSSG, 0.5 M arginine, 10% glycerol) and stirred at 4°C overnight.…”

Expression and Characterization of Natural-Like Recombinant Der p 2 for Sublingual Immunotherapy

“…Other studies reported a successful expression of recombinant Der p 2 in E. coli , alone or as a fusion protein with glutathione-S-transferase or Der p 1 [29,37,49,50,51] as well as in eukaryotic hosts such as Saccharomyces cerevisiae [30,52] and tobacco cells [32]. Some of these molecules were found to resemble the natural counterpart in terms of antigenic properties.…”

“…Small-scale expression was performed as previously described [37]. Briefly, overnight starter cultures were diluted in fresh LB medium and further grown until reaching an OD 600nm of 0.4–0.8.…”

“…Inclusion bodies were recovered and washed as described elsewhere [37]. After solubilisation in 8 M urea, refolding was done by rapid dilution in 50 m M Tris pH 8, 1 m M EDTA, 50 m M NaCl, 5 m M reduced glutathione and 500 m M L -arginine.…”

“…Following SDS-PAGE on 4–12% Bis-Tris acrylamide gels (Invitrogen), Western blots were performed and revealed with either the Dpx-A9 mouse anti-Der p 2 monoclonal antibody (0.2 µg/ml; Indoor Biotechnologies Limited, Warminster, UK) or a pool of sera obtained from HDM-allergic donors, as previously described [37]. Recombinant Bet v 1 (200 ng) [38,39] was used as a negative control.…”