Structural and IgE Binding Analyses of Recombinant Der p 2 Expressed from the Hosts <i>Escherichia coli</i> and <i>Pichia pastoris</i>

Abstract

Background: The house dust mite allergen Der p 2 is one of the most important indoor allergens associated with allergic disease. Recombinant Der (rDer) p 2 with high IgE binding activity can be readily produced in Escherichia coli and Pichia pastoris, but the structure and IgE binding of the different methods of preparation have not been compared. Methods: Secondary structure was assessed by circular dichroism (CD). Intrinsic fluorescence and hydrophobic probe (1-anilinonaphthalene 8-sulphonic acid, ANS) were used to study the Der p 2 hydrophobic cavity. IgE binding was assessed by ELISA inhibition. Results: CD analysis showed the expected secondary structure for both nDer p 2 and refolded Der p 2 prepared from E. coli inclusion bodies but primarily random structure for Der p 2 secreted from P. pastoris. The secreted product, however, had disulphide bonding and could be refolded to a similar structure to natural Der (nDer) p 2 after precipitation with trichloro-acetic or ammonium sulphate. ANS binding and intrinsic Trp92 fluorescence showed that all recombinant proteins were different to nDer p 2 and that the allergen secreted from P. pastoris did not form a hydrophobic cavity. Despite the marked structural changes, all preparations of Der p 2 had similar IgE binding to nDer p 2. Conclusion: Despite almost identical IgE binding, rDer p 2 prepared from both E. coli and P. pastoris showed structural differences to nDer p 2. Der p 2 secreted from P. pastoris lacked most of the natural structure, but refolding could induce the natural structure.

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

“…Altogether, our data demonstrate that the rDer p 2 produced in P. pastoris more closely resembles nDer p 2 when compared to the E. coli -derived rDer p 2. These results differ from those obtained in a recent study indicating that Der p 2 secreted by P. pastoris has a mainly unordered structure [31]. Explanations for such discrepancies are unclear but may originate from differences in yeast strains as well as the expression and purification protocols used.…”

“…Along with other authors, we reported previously that the immunoreactivity and allergenicity of the Der p 2 molecule are still preserved to some extent despite incorrect folding [31,37,51,53], likely due to the presence of linear IgE epitopes [55]. Our results confirm a direct link between correct cysteine pairing and both molecular homogeneity and stability evaluated by resistance to heat denaturation.…”

“…Although Der p 2 has been previously produced in a recombinant form in a variety of expression systems [29,30,31,32], as of today, none of these studies has documented a bona fide natural conformation of the molecule to a level fulfilling the requirements for human administration. As Der p 2 exists as a variety of isoforms/variants [33,34,35], we first selected a prominent isoform following a mass spectrometry (MS) analysis of mite extracts.…”

Effect of Amino Acid Polymorphisms of House Dust Mite Der p 2 Variants on Allergic Sensitization

“…Yeast-expressed recombinant Der p 2 (rDer p 2) was produced as previously described 10 from a single colony of transformed P. pastoris KM71H strain containing Der p 2 variant cDNA and grown in buffered complex media containing glycerol (BMGY) in a ratio of 1:100 at 30°C with shaking until O.D.600 nm of 20.0-25.0. Cells were harvested and resuspended in a solution containing 1% yeast, 2% casamino acid, 1% asparagine, 0.34% Yeast Nitrogen Base, 4×10 -5 % biotin, and 0.5% methanol (YCM) at a 10-fold more concentration and were grown for 72 hours at 30°C.…”

“…10 According to this protocol, 1-anilinonaphthalene 8-sulfonic acid (1,8-ANS) (Sigma, St. Louis, MO, USA) binding to measure the hydrophobic binding capacity of Der p 2 was conducted as before 10 with 1,8-ANS dissolved in methanol at a concentration determined spectrophotometrically using a molar extinction coefficient of 8,000 M/cm at 372 nm. For assay, 3.5 µM natural Der p 2 (nDer p 2) or rDer p 2 variants were mixed with 200 µM ANS for 10 minutes at 25°C.…”