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.
Highlights
Allergy to wasp Vespula vulgaris and Vespula germanica venom is the most common insect sting allergy in temporate Europe and is a cause of significant morbidity, impairment of life quality and can sometimes be fatal [1]
The aim of this study was to express enzymatically inactivated variants of phospholipase A1 from V. vulgaris in methylotrophic yeast P. pastoris, which is well-suited for industrial-scale fermentations due to strain stability, high level of foreign protein secretion, ability to grow to high cell densities on defined minimal medium and low level of secretion of native proteins
On the inspection of the sequence we found that it contained some longer AT-rich stretches, which could serve as premature termination signals to yeast (Figure 1)
Summary
Allergy to wasp Vespula vulgaris and Vespula germanica venom is the most common insect sting allergy in temporate Europe and is a cause of significant morbidity, impairment of life quality and can sometimes be fatal [1]. Patients can be treated by specific immunotherapy (SIT) with venom extract, which is obtained in a tedious and expensive procedure where venom sacs are manually removed from collected wasps and the extract is partially purified. The extract is subject to composition variation, which can cause adverse effects during treatment; it contains a number of non-allergenic proteins [4]. Using recombinant allergens as a vaccine instead of the venom extract could improve the treatment of wasp venom allergies by providing a cheaper, well-characterized, and composition-consistent vaccine. The vaccine components could be combined differently to match individual patients’ sensitization profiles
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