Improved production of the recombinant phospholipase A1 from Polybia paulista wasp venom expressed in bacterial cells for use in routine diagnostics

Abstract

Phospholipase A1 (PLA1) is one of the three major allergens identified in the venom of P. paulista (Hymenoptera: Vespidae), a clinically relevant wasp from southeastern Brazil. The recombinant form of this allergen (rPoly p 1) could be used for the development of molecular diagnostic of venom allergy. Early attempts to produce rPoly p 1 using Escherichia coli BL21 (DE3) cells rendered high yields of the insoluble rPoly p 1 but with low levels of solubilized protein recovery (12%). Here, we aimed to improve the production of rPoly p 1 in E. coli by testing different conditions of expression, solubilization of the inclusion bodies and protein purification. The results showed that the expression at 16 °C and 0.1 mM of IPTG increased the production of rPoly p 1, still in the insoluble form, but with high solubilized protein yields after incubation with citrate-phosphate buffer with 0.15 M NaCl, 6 M urea, pH 2.6 at 25 ºC for 2 h. The venom allergen was also cloned in pPICZαA vector for soluble expression as a secreted protein in Pichia pastoris X-33 cells, rendering almost undetectable levels (nanograms) in the culture supernatant. In contrast, a sevenfold increase of the solubilized and purified rPoly p 1 yields (1.5 g/L of fermentation broth) was obtained after improved production in E. coli. The identity of the protein was confirmed with an anti-His antibody and MS spectra. Allergen-specific IgE (sIgE)-mediated recognition was evaluated in immunoblotting with sera of allergic patients (n = 40). Moreover, rPoly p 1 showed high levels of diagnostic sensitivity (95%). The optimized strategy for rPoly p 1 production described here, will provide the amounts of allergen necessary for the subsequent protein refolding, immunological characterization steps, and ultimately, to the development of molecular diagnostic for P. paulista venom allergy.

Keywords: Molecular diagnostics; Polybia paulista; Recombinant phospholipase A1; Recovery; Solubilization; Venom allergy.

Fig. 1

SDS-PAGE (12%) analyses of the cell lysate insoluble fractions from E. coli BL21 (DE3) obtained after rPoly p 1 expression (16 °C, 10 h post-induction). Samples: N = negative control (non-transformed E. coli BL21 (DE3) induced with 1 mM IPTG, C = positive control (rPoly p 1 expression under previously condition tested (Perez-Riverol et al. 2016)) and 1–0.005 = transformed E. coli BL21 (DE3) cells after induction with 1, 0.5, 0.1, 0.05, 0.01 and 0.005 mM IPTG. MW = molecular weight marker

Fig. 2

SDS-PAGE (12%) of the insoluble (I) and soluble (S) fractions obtained after protein solubilization of E. coli cell lysate pellets in presence of 8 M urea, at 25 °C. Buffers: B1 = Citrate–phosphate, B2 = Tris–HCl, B3 = Phosphate and B4 = Carbonate-bicarbonate. MW molecular weight marker

Fig. 3

SDS-PAGE (12%) of the insoluble (I) and soluble (S) fractions obtained after protein solubilization of E. coli cell lysate pellets in citrate phosphate buffer pH 2.6 (B1), containing 3 M, 4 M and 6 M urea, at 25 °C. MW Molecular weight marker

Fig. 4

SDS-PAGE (12%) of (a) and 6XHis-mediated immunodetection (b) of the purified rPoly p 1. Samples: Venom = crude venom extract of P. paulista, Nat = nPoly p 1, Rec = rPoly p 1. MW Molecular weight markers are indicated

Fig. 5

Sequence of the recombinant form of P. paulista venom phospholipase A1 obtained from the interpretation of the CID spectra of the peptides generated by digestion with: Asp-N protease (horizontal filled bar), trypsin (horizontal empty bar), chymotrypsin (horizontal grey bar) and protease Glu-C/V8 (horizontal dark grey bar)

Fig. 6

Recombinant Poly p 1 cloning and expression in yeast. (a) Positive P. pastoris transformants detecting by PCR, using primers 5´AOX1 and 3´AOX1. (b) SDS-PAGE (12%) and (c) anti-His immunoblotting analyses of the supernatant-collected fractions at 24, 48, 72, 96, 120 h post induction. Samples: P = pPICZαA_poly p 1 construct as positive control, N = P. pastoris transformed with empty pPICZαA; 1–9 clones. Molecular weight markers are indicated

Fig. 7

IgE-mediated immunodetection of rPoly p 1 expressed in E. coli by sera of allergic patients sensitized to wasp venom. Samples: 1–10 = individual serum, N = randomly selected serum from a non-allergic healthy volunteers, P = Pool of sera (5) from sensitized individuals. MW molecular weight marker. Bands were visualized in an Image Quant 400 (GE Healthcare, Sweden) using the chemiluminescent Luminata™ ForteWestern HRP substrate (Millipore, MA, US)