Molecular cloning and expression in Pichia pastoris of a hypoallergenic antigen 5.
Stings by insects from the Hymenoptera order can cause life-threatening allergic reactions and impair life quality. Immunotherapy with venom extracts is the most extensively employed treatment to reduce morbidity and mortality, but purified and safer allergy vaccines are needed. Antigen 5 is an important allergen of vespid venoms. We previously reported that Antigen 5 from Polybia scutellaris (Poly s 5) is likely to be a hypoallergenic variant. On the basis of such findings, this work deals with the recombinant expression and purification of Poly s 5 in Pichia pastoris. In order to overcome non-native glycosylation of the recombinant protein, it was necessary to delete a glycosylation site. On the other hand, different strategies were attempted to obtain a satisfactory yield of the protein; moreover, the influence of the methanol concentration in the expression medium was investigated and found to be crucial. Mass spectrometry, N-terminal sequencing, and IgG-binding inhibition assays were performed. Results allowed us to confirm the immunological equivalence between the recombinant and the natural proteins. In conclusion, a novel protocol for the recombinant expression of Poly s 5 in P. pastoris was designed thus bringing about a high yield of the protein useful for clinical and scientific purposes.
Vinzón
Sabrina E
SE
Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Instituto de Química y Fisicoquímica Biológicas, Junín 956 (1113) Buenos Aires, Argentina.
Pirpignani
María L
ML
Nowicki
Cristina
C
Biscoglio de Jiménez Bonino
Mirtha
M
D000818:Animals; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006031:Glycosylation; D057166:High-Throughput Screening Assays; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D021241:Spectrometry, Mass, Electrospray Ionization; D014862:Wasp Venoms / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology
D011994:Recombinant Proteins; D014862:Wasp Venoms; C469232:antigen 5, Polybia scutellaris