Expression of Enzymatically Inactive Wasp Venom Phospholipase A1 in Pichia pastoris

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.

Molecular cloning, expression and IgE-immunoreactivity of phospholipase A1, a major allergen from Polybia paulista (Hymenoptera: Vespidae) venom

“…(À) corresponds to a negative control (non-induced clone at T 10 ). expressed in E. coli (King et al, 1997;Skov et al, 2006;Lockwood et al, 2012) and other systems (Vinz on et al, 2010;Borodina et al, 2011). This agrees with the feasibility of the cell system and the purification procedure employed to express rPoly p 1 at high rates.…”

“…Finally, other expression platforms could be used to obtain rPoly p 1 as a soluble protein. For instance, the expression of Ves v 5 on yeast (Borodina et al, 2011) and insect cells (Seismann et al, 2010) circumvented insolubility problems of heterologous allergen production. Previous attempts to obtain PLA1s from the venom of social Hymenoptera in E. coli also resulted in their expression as insoluble proteins.…”

Characterization of Venom Components and Their Phylogenetic Properties in Some Aculeate Bumblebees and Wasps

“…Phospholipase A2 was also highly expressed in most social wasps, exhibiting TPM value > 600, especially in V. crabro, V. dybowskii, and V. simillima (Figure 1). As phospholipase A1 and A2 are major Hymenopteran venom allergens in humans [33,34], their high expression in the venoms social wasps suggests that they are likely to be involved in the defensive function of venom.…”

Facing Hymenoptera Venom Allergy: From Natural to Recombinant Allergens

“…To date, however, a limited number of Hymenoptera venom allergens have been expressed using this yeast. For instance, high levels of a non-enzymatically active form of Ves v 1 were produced in P. pastoris [ 96 ] after introducing a point mutation at the active site of the allergen. The modified r Ves v 1 form was produced as a secreted soluble protein facilitating the subsequent purification steps.…”