Improved production of the recombinant phospholipase A1 from wasp venom expressed in bacterial cells for use in routine diagnostics.
Amilcar Perez-Riverol, Alexis Musacchio-Lasa, Luis Gustavo Romani Fernandes, Jose Roberto Aparecido Dos Santos-Pinto, Franciele Grego Esteves, Murilo Luiz Bazon, Ricardo de Lima Zollner, Mario Sergio Palma, Márcia Regina Brochetto-Braga
Author Information
Amilcar Perez-Riverol: 1Center for the Study of Social Insects, Department of General and Applied Biology, University of Sao Paulo State (UNESP/RC), São Paulo, Brazil.
Alexis Musacchio-Lasa: 2Center for Genetic Engineering and Biotechnology. Biomedical Research Division, System Biology Department, Ave. 31, e/ 158 and 190, Cubanacan, Playa, P.O. Box 6162, 10600 Havana, Cuba.
Luis Gustavo Romani Fernandes: 3Laboratory of Translational Immunology, School of Medical Sciences, University of Campinas (UNICAMP), Campinas, São Paulo, Brazil.
Jose Roberto Aparecido Dos Santos-Pinto: 1Center for the Study of Social Insects, Department of General and Applied Biology, University of Sao Paulo State (UNESP/RC), São Paulo, Brazil.
Franciele Grego Esteves: 1Center for the Study of Social Insects, Department of General and Applied Biology, University of Sao Paulo State (UNESP/RC), São Paulo, Brazil.
Murilo Luiz Bazon: 4Arthropod Molecular Biology Laboratory-LBMA-IB, University of Sao Paulo State (UNESP/RC), Ave. 24-A, 1515- Bela Vista, Rio Claro, SP 13506-900 Brazil.
Ricardo de Lima Zollner: 3Laboratory of Translational Immunology, School of Medical Sciences, University of Campinas (UNICAMP), Campinas, São Paulo, Brazil.
Mario Sergio Palma: 1Center for the Study of Social Insects, Department of General and Applied Biology, University of Sao Paulo State (UNESP/RC), São Paulo, Brazil.
Márcia Regina Brochetto-Braga: 4Arthropod Molecular Biology Laboratory-LBMA-IB, University of Sao Paulo State (UNESP/RC), Ave. 24-A, 1515- Bela Vista, Rio Claro, SP 13506-900 Brazil. ORCID
Phospholipase A1 (PLA1) is one of the three major allergens identified in the venom of (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 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 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 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 . 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 ( = 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 venom allergy.
