Heterologous Expression, Purification and Immunoreactivity of the Antigen 5 from Polybia paulista Wasp Venom

Abstract

Polybia paulista (Hymenoptera: Vespidae) is responsible for a high number of sting accidents and anaphylaxis events in Southeast Brazil, Argentina and Paraguay. The specific detection of allergy to the venom of this wasp is often hampered by the lack of recombinant allergens currently available for molecular diagnosis. Antigen 5 (~23 kDa) from P. paulista venom (Poly p 5) is a highly abundant and glycosylated allergenic protein that could be used for development of component-resolved diagnosis (CRD). Here, we describe the cloning and heterologous expression of the antigen 5 (rPoly p 5) from P. paulista venom using the eukaryotic system Pichia pastoris. The expression as a secreted protein yielded high levels of soluble rPoly p 5. The recombinant allergen was further purified to homogeneity (99%) using a two-step chromatographic procedure. Simultaneously, the native form of the allergen (nPoly p 5) was purified from the wasp venom by Ion exchange chromatography. The rPoly p 5 and nPoly p 5 were then submitted to a comparative analysis of IgE-mediated immunodetection using sera from patients previously diagnosed with sensitization to wasp venoms. Both rPoly p 5 and nPoly p 5 were recognized by specific IgE (sIgE) in the sera of the allergic individuals. The high levels of identity found between nPoly p 5 and rPoly p 5 by the alignment of its primary sequences as well as by 3-D models support the results obtained in the immunoblot. Overall, we showed that P. pastoris is a suitable system for production of soluble rPoly p 5 and that the recombinant allergen represents a potential candidate for molecular diagnosis of P.paulista venom allergy.

Keywords: Polybia paulista; allergy; antigen 5; diagnosis; heterologous expression.

Figure 1

Nucleotide and predicted amino acid sequences of rPoly p 5 (Gen Bank: ANW82807.1) Forward (red arrow) and reverse (green arrow) primers used for gene specific amplification are indicated. Cysteine residues potentially involved in disulfide bridges (discontinuous red lines) are highlighted in yellow and the position marked with red triangles. The consensus sequons for N-glycosylation are in light blue with the asparagine residue enclosed in black ovals. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Figure 2

Multiple alignment and identity values among the primary sequence of the predicted rPoly p 5 protein (GenBank: ANW82807.1) with the primary sequences of nPoly p 5 (Polybia paulista), nPoly s 5 (Polybia scutellaris rioplatensis), nVes v 5 (Vespula vulgaris), nPol d 5 (Polistes dominula), nPol a 5 (Polistes annularis), and nSol i 3 (Solenopsis invicta). The most important regions of identity are marked in dark gray, highlighting the highly conserved residues in this type of alignment.

Figure 3

SDS-PAGE (12%) of the supernatant from P. pastoris X-33 culture during rPoly p 5 expression. Abbreviations: 24–120, hours after induction (1% methanol); e, negative control (P. pastoris X-33 transformed with an empty pPICZαA vector).

Figure 4

SDS-PAGE (12%) analysis of fractions eluted at 75 mM during rPoly p 5 purification by IMAC. E, Elution.

Figure 5

Sephadex G-100 size-exclusion chromatography profile of the sample eluted from Ni²⁺ Sepharose affinity chromatography (a). The chromatography was conducted with 5 mM ammonium acetate pH 6.8 buffer and 1 mL fractions were collected at a flow rate of 1 mL/min. Protein elution was monitored at 280 nm (___). The band profile in (12%) SDS-PAGE of the most representative fraction (numbered and marked with black triangles) is shown (b) along with the immunodetection of the purified rPoly p 5 using anti-His antibody (c).

Figure 6

Purification of native Poly p 5 from P. paulista venom. Cation-exchange chromatography profile obtained after crude venom extract fractionation, using a Hiprep FF CM column (16 × 10 mm, 20 mL; GE Healthcare) coupled to an AKTA-FPLC system, similar to previously obtained [12,26]. The elution was performed under a linear gradient from 0 to 1 M NaCl (_ _ _). The protein was monitored by measuring the absorbance at 280 nm, represented by a continuous line (___) (a). Two-milliliter fractions were collected for further detection of nPoly p 5 (~25 kDa) by 12% SDS-PAGE of all fractions eluted, and the gels stained with silver stain. A representation of the SDS-PAGE profile showing a unique band of ~25 kDa obtained from the main fractions (20–25) of the peak V (b). The crude venom (B), the purified native (n) and recombinant (r) Poly p 5 were finally analyzed in a (12%) SDS-PAGE (c).

Figure 7

IgE-mediated immunodetection of native (a) and recombinant (b) Poly p 5. Serum samples: P1-P10 = individual sera from patients previously diagnosed with specific IgE to P. paulista venom; N = pool of five serum samples from nonsensitized healthy volunteers, as negative control. The molecular weight marker (kDa) is indicated. The results of (a,b) are supported by the in silico analyses of the 3-D models of nPoly p 5 and rPoly p 5 showing the high structural homology between them (c) which is predicted by the presence of commons linear and conformational epitopes. The root-mean-square deviation (RMDS) of atomic positions of the 3-D models from nPoly p 5 and rPoly p 5 compared to the template 3-D model (Ves v 5, PDB 1QNX) is showed in the blue box.