Display of wasp venom allergens on the cell surface of Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>Yeast surface display is a technique, where the proteins of interest are expressed as fusions with yeast surface proteins and thus remain attached to the yeast cell wall after expression. Our purpose was to study whether allergens expressed on the cell surface of baker's yeast <it>Saccharomyces cerevisiae </it>preserve their native allergenic properties and whether the yeast native surface glycoproteins interfere with IgE binding. We chose to use the major allergens from the common wasp <it>Vespula vulgaris </it>venom: phospholipase A1, hyaluronidase and antigen 5 as the model.</p> <p>Results</p> <p>The proteins were expressed on the surface as fusions with a-agglutinin complex protein AGA2. The expression was confirmed by fluorescent cytometry (FACS) after staining the cells with antibody against a C-tag attached to the C-terminal end of the allergens. Phospholipase A1 and hyaluronidase retained their enzymatic activities. Phospholipase A1 severely inhibited the growth of the yeast cells. Antigen 5 - expressing yeast cells bound IgE antibodies from wasp venom allergic patient sera but not from control sera as demonstrated by FACS. Moreover, antigen 5 - expressing yeast cells were capable of mediating allergen-specific histamine release from human basophils.</p> <p>Conclusions</p> <p>All the three major wasp venom allergens were expressed on the yeast surface. A high-level expression, which was observed only for antigen 5, was needed for detection of IgE binding by FACS and for induction of histamine release. The non-modified <it>S. cerevisiae </it>cells did not cause any unspecific reaction in FACS or histamine release assay despite the expression of high-mannose oligosaccharides.</p> <p>In perspective the yeast surface display may be used for allergen discovery from cDNA libraries and possibly for sublingual immunotherapy as the cells can serve as good adjuvant and can be produced in large amounts at a low price.</p

Expression of enzymatically inactive wasp venom phospholipase A1 in Pichia pastoris

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 up to the level of 4 mg/L in shake flask culture. It 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

Biochemical and kinetic characterisation of a novel xylooligosaccharide-upregulated GH43 β-d-xylosidase/α-l-arabinofuranosidase (BXA43) from the probiotic Bifidobacterium animalis subsp. lactis BB-12

The Bifidobacterium animalis subsp. lactis BB-12 gene BIF_00092, assigned to encode a β-d-xylosidase (BXA43) of glycoside hydrolase family 43 (GH43), was cloned with a C-terminal His-tag and expressed in Escherichia coli. BXA43 was purified to homogeneity from the cell lysate and found to be a dual-specificity exo-hydrolase active on para-nitrophenyl-β-d-xylopyranoside (pNPX), para-nitrophenyl-α-L-arabinofuranoside (pNPA), β-(1 → 4)-xylopyranosyl oligomers (XOS) of degree of polymerisation (DP) 2–4, and birchwood xylan. A phylogenetic tree of the 92 characterised GH43 enzymes displayed five distinct groups (I − V) showing specificity differences. BXA43 belonged to group IV and had an activity ratio for pNPA:pNPX of 1:25. BXA43 was stable below 40°C and at pH 4.0–8.0 and showed maximum activity at pH 5.5 and 50°C. K(m) and k(cat) for pNPX were 15.6 ± 4.2 mM and 60.6 ± 10.8 s(-1), respectively, and substrate inhibition became apparent above 18 mM pNPX. Similar kinetic parameters and catalytic efficiency values were reported for β-d-xylosidase (XynB3) from Geobacillus stearothermophilus T‒6 also belonging to group IV. The activity of BXA43 for xylooligosaccharides increased with the size and was 2.3 and 5.6 fold higher, respectively for xylobiose and xylotetraose compared to pNPX. BXA43 showed clearly metal inhibition for Zn(2+) and Ag(+), which is different to its close homologues. Multiple sequence alignment and homology modelling indicated that Arg(505)Tyr(506) present in BXA43 are probably important for binding to xylotetraose at subsite +3 and occur only in GH43 from the Bifidobacterium genus

Effect of Particle Morphology on the Ripening of Supported Pt Nanoparticles

To improve the understanding of sintering in diesel and lean-burn engine exhaust aftertreatment catalysts, we examined oxygen-induced sintering in a model catalyst consisting of Pt nanoparticles supported on a planar, amorphous Al2O3 substrate. After ageing at increasing temperatures, transmission electron microscopy analysis reveals that highly monodisperse ensembles of nanoparticles transformed into ensembles with bimodal and subsequently Lifshitz-Slyozov-Wagner particle size distributions. Moreover, scanning transmission electron microscopy and atomic force microscopy analysis suggest that the Pt nanoparticles have size-dependent morphologies after sintering in oxidizing environment. The evolution of the particle sizes is described by a simple kinetic model for ripening and the size-dependent particle morphology is proposed as an explanation for the observed bimodal particle size distribution shapes