| Full text | |
| Author(s): |
Perez-Riverol, Amilcar
[1]
;
Musacchio-Lasa, Alexis
[2]
;
Romani Fernandes, Luis Gustavo
[3]
;
Aparecido dos Santos-Pinto, Jose Roberto
[1]
;
Esteves, Franciele Grego
[1]
;
Bazon, Murilo Luiz
[4]
;
Zollner, Ricardo de Lima
[3]
;
Palma, Mario Sergio
[1]
;
Brochetto-Braga, Marcia Regina
[4, 5]
Total Authors: 9
|
| Affiliation: | [1] Univ Sao Paulo State UNESP RC, Dept Gen & Appl Biol, Ctr Study Social Insects, Sao Paulo - Brazil
[2] Ctr Genet Engn & Biotechnol, Syst Biol Dept, Biomed Res Div, Ave 31, E-158 & 190, POB 6162, Havana 10600 - Cuba
[3] Univ Campinas UNICAMP, Sch Med Sci, Lab Translat Immunol, Campinas, SP - Brazil
[4] Univ Sao Paulo State UNESP RC, Arthropod Mol Biol Lab LBMA IB, Ave 24-A, 1515 Bela Vista, Rio Claro 13506900, SP - Brazil
[5] Univ Sao Paulo State UNESP, Venoms & Venomous Anim Studies Ctr CEVAP, Lageado Expt Farm, Jose Barbosa de Barros St 1780, Botucatu 18610307, SP - Brazil
Total Affiliations: 5
|
| Document type: | Journal article |
| Source: | 3 BIOTECH; v. 10, n. 5 APR 27 2020. |
| Web of Science Citations: | 0 |
| 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 degrees 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 pPICZ alpha A 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. (AU) | |
| FAPESP's process: | 14/13936-7 - Allergens from venom of the social wasp Polybia paulista (Hymenoptera, Vespidae): recombinant expression in yeast for improved diagnosis and specific immunotherapy to Hymenoptera venom allergy |
| Grantee: | Márcia Regina Brochetto Braga |
| Support Opportunities: | Regular Research Grants |
| FAPESP's process: | 13/26451-9 - Bioprospecting and Structural Analysis of the Silk Proteins of Arthropods by a Proteomics Approach Using nanoLC-ESI-CID/ETD System |
| Grantee: | José Roberto Aparecido dos Santos-Pinto |
| Support Opportunities: | Scholarships in Brazil - Post-Doctorate |
| FAPESP's process: | 19/02298-3 - Characterization of the immunological potential of the recombinant antigen 5 from Polybia paulista venom |
| Grantee: | Murilo Luiz Bazon |
| Support Opportunities: | Scholarships abroad - Research Internship - Doctorate |
| FAPESP's process: | 17/10373-0 - Profiling the peptidomic and structural-functional characterization of lipid vesicles present in the Nephila clavipes web spider |
| Grantee: | Franciele Grego Esteves |
| Support Opportunities: | Scholarships in Brazil - Doctorate |
| FAPESP's process: | 15/14220-8 - Proteometabolomic characterization of the spider web components Nephila clavipes used in prey capture strategy |
| Grantee: | Franciele Grego Esteves |
| Support Opportunities: | Scholarships in Brazil - Master |
| FAPESP's process: | 17/07988-2 - Biological and inflammatory evaluation of the antigen 5 from the venom of Polybia paulista (Hymenoptera, Vespidae) in the recombinant form, on cells of macrophagic lineage |
| Grantee: | Murilo Luiz Bazon |
| Support Opportunities: | Scholarships in Brazil - Doctorate |