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.
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Abbreviations
- PLA1:
-
Phospholipase A1
- Poly p 1:
-
Phospholipase A1 from Polybia paulista venom
- Poly p 5:
-
Antigen 5 from Polybia paulista venom
- CCDs:
-
Cross-reactive carbohydrate determinants
- HVA:
-
Hymenoptera venom allergy
- HBV:
-
Honeybee venom
- CRD:
-
Component resolved diagnosis
- sIgE:
-
Specific IgE
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Acknowledgements
The authors acknowledge the financial support from FAPESP (Sao Paulo Research Foundation) (Proc.# 2014/13936-7), CAPES (Coordination for the Improvement of Higher Education Personnel-Brazil), CNPq (National Council for Scientific and Technological) and Postgraduate Program of Biological Sciences (Cellular and Molecular Biology) at UNESP, Rio Claro, SP, Brazil. APR thanks CAPES-DS for the Doctoral-Grant (#1257664). JRASP (FAPESP #2013/26451-9 and Portaria Capes 206 de 2018. FGE (FAPESP #2015/14220-8 and #2017/10373-0). MLB thanks FAPESP (#2017/07988-2, # 2019/02298-3 for his Doctoral scholarship) and (CNPq # 455422/2014-1). This study was financed also in part by CAPES) Finance Code 001.
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All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by PRA, SPJRA, EFG, RFLG and BML. The first draft of the manuscript was written by PRA and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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Perez-Riverol, A., Musacchio-Lasa, A., Fernandes, L.G.R. et al. Improved production of the recombinant phospholipase A1 from Polybia paulista wasp venom expressed in bacterial cells for use in routine diagnostics. 3 Biotech 10, 217 (2020). https://doi.org/10.1007/s13205-020-02202-8
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DOI: https://doi.org/10.1007/s13205-020-02202-8