IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
artículos
Título:
Molecular cloning and expression in Pichia pastoris of a hypoallergenic Antigen 5
Autor/es:
SABRINA VINZÓN, NOELIA MASSARI, ELENA RIVERA AND MIRTHA BISCOGLIO DE JIMÉNEZ BONINO
Revista:
PROTEIN EXPRESSION AND PURIFICATION
Editorial:
ACADEMIC PRESS INC ELSEVIER SCIENCE
Referencias:
Año: 2010 vol. 73 p. 23 - 30
ISSN:
1046-5928
Resumen:
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 Pichia pastoris was designed thus bringing about a high yield of the protein useful for clinical and scientific purposes.