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.