Expression of Enzymatically Inactive Wasp Venom Phospholipase A1 in Pichia pastoris
Open Access
- 23 June 2011
- journal article
- research article
- Published by Public Library of Science (PLoS) in PLOS ONE
- Vol. 6 (6), e21267
- https://doi.org/10.1371/journal.pone.0021267
Abstract
Wasp venom allergy is the most common insect venom allergy in Europe. It is manifested by large local reaction or anaphylactic shock occurring after a wasp sting. The allergy can be treated by specific immunotherapy with whole venom extracts. Wasp venom is difficult and costly to obtain and is a subject to composition variation, therefore it can be advantageous to substitute it with a cocktail of recombinant allergens. One of the major venom allergens is phospholipase A1, which so far has been expressed in Escherichia coli and in insect cells. Our aim was to produce the protein in secreted form in yeast Pichia pastoris, which can give high yields of correctly folded protein on defined minimal medium and secretes relatively few native proteins simplifying purification. Residual amounts of enzymatically active phospholipase A1 could be expressed, but the venom protein had a deleterious effect on growth of the yeast cells. To overcome the problem we introduced three different point mutations at the critical points of the active site, where serine137, aspartate165 or histidine229 were replaced by alanine (S137A, D165A and H229A). All the three mutated forms could be expressed in P. pastoris. The H229A mutant did not have any detectable phospholipase A1 activity and was secreted at the level of several mg/L in shake flask culture. The protein was purified by nickel-affinity chromatography and its identity was confirmed by MALDI-TOF mass spectrometry. The protein could bind IgE antibodies from wasp venom allergic patients and could inhibit the binding of wasp venom to IgE antibodies specific for phospholipase A1 as shown by Enzyme Allergo-Sorbent Test (EAST). Moreover, the recombinant protein was allergenic in a biological assay as demonstrated by its capability to induce histamine release of wasp venom-sensitive basophils. The recombinant phospholipase A1 presents a good candidate for wasp venom immunotherapy.Keywords
This publication has 33 references indexed in Scilit:
- Display of wasp venom allergens on the cell surface of Saccharomyces cerevisiaeMicrobial Cell Factories, 2010
- Recombinant phospholipase A1 (Ves v 1) from yellow jacket venom for improved diagnosis of hymenoptera venom hypersensitivityClinical and Molecular Allergy, 2010
- Dissecting cross-reactivity in hymenoptera venom allergy by circumvention of α-1,3-core fucosylationMolecular Immunology, 2009
- Structural and immunological characterization of the N-glycans from the major yellow jacket allergen Ves v 2: The N-glycan structures are needed for the human antibody recognitionMolecular Immunology, 2009
- Affinity of IgE and IgG against cross-reactive carbohydrate determinants on plant and insect glycoproteinsJournal of Allergy and Clinical Immunology, 2007
- In vitro hymenoptera venom allergy diagnosis: improved by screening for cross‐reactive carbohydrate determinants and reciprocal inhibitionAllergy, 2006
- Allergen-specific immunotherapy with recombinant grass pollen allergensJournal of Allergy and Clinical Immunology, 2005
- Insect sting allergy and venom immunotherapy: A model and a mysteryJournal of Allergy and Clinical Immunology, 2005
- Allergen‐specific immunosuppression by mucosal treatment with recombinant Ves v 5, a major allergen of Vespula vulgaris venom, in a murine model of wasp venom allergyImmunology, 2003
- A new method for detecting histamine releaseInflammation Research, 1984