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.
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ABSTRACT: Phospholipase A(1) (PLA(1)) has been described in the infective stages of Trypanosoma cruzi as a membrane-bound/secreted enzyme that significantly modified host cell lipid profile with generation of second lipid messengers and concomitant activation of Protein Kinase C. In the present work we determined higher levels of PLA(1) expression in the infective amastigotes and trypomastigotes than in the non-infective epimastigotes of lethal RA strain. In addition, we found similar expression patterns but distinct PLA(1) activity levels in bloodstream trypomastigotes from Cvd and RA (lethal) and K98 (non-lethal) T. cruzi strains, obtained at their corresponding parasitemia peaks. This fact was likely due to the presence of different levels of anti-T. cruzi PLA(1) antibodies in sera of infected mice, that modulated the enzyme activity. Moreover, these antibodies significantly reduced in vitro parasite invasion indicating the participation of T. cruzi PLA(1) in the early events of parasite-host cell interaction. We also demonstrated the presence of Lysophospholipase activity in live infective stages that could account for self-protection against the toxic lysophospholipids generated by T. cruzi PLA(1) action. At the genome level, we identified at least eight putative genes that codify for T. cruzi PLA(1) with high amino acid sequence variability in their amino and carboxy-terminal regions; a putative PLA(1) selected gene was cloned and expressed as a recombinant protein that possessed PLA(1) activity. Collectively, the results presented here point out at T. cruzi PLA(1) as a novel virulence factor implicated in parasite invasion.Molecular and Biochemical Parasitology 12/2012; · 2.73 Impact Factor
Expression of Enzymatically Inactive Wasp Venom
Phospholipase A1 in Pichia pastoris
Irina Borodina1, Bettina M. Jensen2, Tim Wagner1, Maher A. Hachem3, Ib Søndergaard1, Lars K.
1Center for Microbial Biotechnology, Institute of Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark, 2Allergy Clinic, Dermato-Allergological Dept.
K, CUH-Gentofte, Rigshospitalet Dept 7551, København Ø, Denmark, 3Enzyme and Protein Center, Institute of Systems Biology, Technical University of Denmark, Kgs.
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.
Citation: Borodina I, Jensen BM, Wagner T, Hachem MA, Søndergaard I, et al. (2011) Expression of Enzymatically Inactive Wasp Venom Phospholipase A1 in Pichia
pastoris. PLoS ONE 6(6): e21267. doi:10.1371/journal.pone.0021267
Editor: Alfredo Herrera-Estrella, Cinvestav, Mexico
Received February 16, 2011; Accepted May 25, 2011; Published June 23, 2011
Copyright: ? 2011 Borodina et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by Grant 274-07-0148 from the Danish Research Council. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
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 . Epidemiologic studies report prevalence of
systemic anaphylactic reactions in general population at 1–1.5%
[2,3]. 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 then the extract is partially purified. The
extract is subject to composition variation, which can cause
adverse effects during treatment; furthermore it contains a number
of non-allergenic proteins . Using recombinant allergens as a
vaccine instead of the venom extract could improve the treatment
of wasp venom allergies by providing a cheaper, well-character-
ized, and composition-consistent vaccine. Additionally the vaccine
components could be combined differently to match individual
patients’ sensitization profiles.
One V. vulgaris sting injects around 1.7–3.1 mg of venom, in
which the most abundant allergenic proteins (major allergens) are
phospholipase A1 (Ves v 1.0101), hyaluronidase (Ves v 2.0101)
and antigen 5 (Ves v 5.0101), accounting for correspondingly
3.3%, 1.5% and 8.1% of the total venom protein . A close
homologue to hyaluronidase, though without enzymatic activity,
allergen Ves v 2.0201 has been found [6,7]. Recently IgE
reactivity and basophils activation has also been shown for a high-
molecular mass venom component, 100 kDa dipeptidyl peptidase
IV (Ves v 3.0101) . Allergens from V. vulgaris have been
recombinantly expressed in various hosts as E. coli, insect cells and
Antigen 5, a 23 kDa non-glycosylated protein with so far
unknown function, has been expressed in E. coli [9,10], P. pastoris
, insect cells  and recently also on the surface of yeast
Saccharomyces cerevisiae . Antigen 5 produced in P. pastoris has
recently become commercially available for diagnostic purposes in
ImmunoCAP format (Phadia, Sweden).
Hyaluronidase, 45-kDa glycosylated protein, catalyzing hya-
luronic acid degradation and thus facilitating spreading of venom
components in the tissue after sting, has been expressed in E. coli
[13,14] and in insect cells . The protein expressed in E. coli did
not obtain enzymatic activity after refolding procedure  and
PLoS ONE | www.plosone.org1 June 2011 | Volume 6 | Issue 6 | e21267
had a lower reactivity towards antibodies specific for the native
hyaluronidase, indicating that parts of the discontinuous epitopes
were lost due to improper folding . It has been hypothesized
that glycosylation is important for enzymatic activity and possibly
also for correct folding of hyaluronidase . The importance of
hyaluronidase for allergic response to wasp venom is probably low
as Ves v 2 - specific antibodies are mainly directed towards cross-
reactive carbohydrate determinates [15,17], which are believed to
be of low (if any) clinical significance .
Phospholipase A1, a 33.4 kDa non-glycosylated protein,
removes the 1stacyl group from phospholipids and thus causes
damage to cell membranes. Phospholipase A1, expressed in E. coli
had a lower binding to antibodies specific for the native
phospholipase A1 than the native phospholipase A1, suggesting
that the recombinant phospholipase A1 was not correctly folded
. Enzymatically active and an inactivated variant with two
mutations in the putative active site (S137G and D165A) have
been expressed in insect cells, both variants were biologically
While insect cells can provide allergens useful for diagnostic tests
[11,19], the system is less suited for making proteins for
therapeutic applications because of low yields, difficulties with
scale-up, complex purification process and legal issues. In spite of
the long history of baculovirus expression system, only one
baculovirus-derived product has been approved by Federal Drug
Administration (FDA) so far, namely Cervarix, manufactured by
GlaxoSmithKline (UK) . An alternative expression system for
inexpensive protein secretion is yeast, where particularly P. pastoris
has been extensively used recently with several products in the
clinical trials pipeline [21,22] and one FDA-approved product –
Kalbitor (Dyax, USA) .
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 fermenta-
tions 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.
Materials and Methods
The Ethical Committee for the Capital Region of Denmark
approved the use of historical blood samples in the project, since
the patients had already given their informed consent to the
storage and scientific use of their serum samples when their blood
was originally drawn. The informed consent procedure was
written and verbal. At their first visit to the clinic, the patient is
informed that if he or she consents the sample will be stored for
possible future analyses relating to his/her treatment and for
possible research and development. In case of acceptance by the
patient, an informed consent form is signed by the patient, in
which it is stated that the patient accept that his/her sample is
stored for up to 10 years for these purposes only, and that he/she
at any time can have the sample removed from the serum bank.
Twenty two patients were chosen based on their serum IgE
reactivity with venom extract (i3) in ImmunoCap assay (Phadia),
all CAP class 4 or above, and non-detected cross-reactivity with
honey bee venom (i1, ,0.35 kUa/L). The negative control sera
consisted of a pool of 200 non-allergic sera (negative for common
inhalation allergens (birch, grass, mugwort, horse, dog, cat, house
dust mites and molds) and food allergens (milk, egg white, cod,
peanut, soy bean, wheat flour).
The chemicals were purchased from Sigma-Aldrich and BD
Biosciences. ZeocinTMwas purchased from Invitrogen. The Anti-
His horse radish peroxidase conjugated antibody was a mouse
monoclonal IgG1 antibody (Qiagen). The restriction enzymes and
T4 DNA ligase were purchased from New England Biolabs Inc.
Vespula venom extract (1,000 SQU/ml) used in ELISA was a kind
gift of Jørgen Nedergaard Larsen (ALK Abello ´, Hørsholm,
Denmark). Vespula venom extract used in histamine release assay
was from ALK Abello ´ and contained 136 mg/ml protein. The
primers were ordered from Eurofins MWG Operon (Germany).
Strains and plasmids
The E. coli strain used for cloning was DH5a (F2W80lacZDM15
D(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rk2, mk+) phoA-
supE44 thi-1 gyrA96 relA1 l-). The P. pastoris strains used for protein
expression were X33 (Mut+) and KM71H (MutS, arg4 aox1::ARG4)
(Invitrogen). Vector pPICZalphaA was also purchased from
Invitrogen. For longer storage the E. coli strains were stored in
LB medium with 25% glycerol and yeast strains were stored in
YPD (1% yeast extract, 2% peptone, 2% dextrose) with 15%
glycerol at 280uC.
Cloning and mutation of phospholipase A1 gene
The Vesv1 gene encoding phospholipase A1 was previously
cloned from local Danish V. vulgaris insects . The gene was
codon-optimized using online tool from Mr.Gene GmbH
(Germany) and synthesized by the same company. The gene was
amplified with vesv1_fw and vesv1_rv primers, the fragment was
purified from the 1% agarose gel, digested with XhoI and XbaI and
ligated into pPICZaA plasmid, digested with the same enzymes
and gel-purified. The ligation mixture was transformed into E. coli
DH5a cells and the transformants were selected on low salt Luria-
Bertani (LB) medium with 25 mg/ml zeocin. The presence of the
insert in the plasmid was tested by colony PCR and the correct
transformants were grown overnight in liquid low-salt LB medium
with zeocin selection after which the plasmids were isolated. The
correct cloning of the Vesv1 gene was confirmed by restriction
analysis and sequencing (StarSEQ, Germany).
The plasmid was mutated by site-directed mutagenesis using
QuickChangeH II XL Site-Directed Mutagenesis kit from
Stratagene (USA). The primers used in pairs to generate three
mutations are given in Table 1.
The 4 constructed plasmids were linearized with SacI and
transformed into P. pastoris strains X33 and KM71H using the
optimized electroporation protocol by Wu and Letchworth .
The transformants were selected on YPDS medium (same as YPD,
but with additionally 1 M sorbitol) with 100 mg/ml zeocin and
streak-purified. The integration of the plasmids in the yeast
genome was confirmed by colony PCR.
Cultivation and induction of yeast cells
For X33 Mut+strains a single colony was inoculated into 50 ml
BMGY (buffered minimal glycerol medium, recipe from Invitro-
gen) in 500-ml baffled shake flask and grown for 16 hours at 30uC
with shaking at 150 rpm. The OD600of the culture was measured
and a suitable volume was centrifuged to give on resuspension in
BMMY (buffered minimal methanol medium) an OD600of 1. The
BMMY medium contained 1% methanol and additionally 1%
casamino acids. 25 ml of cells resuspended in BMMY was
transferred to a 500-ml baffled shake flask and induction was
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carried out either at 20uC or 30uC at 150 rpm rotation for
72 hours with daily addition of 1% methanol.
For MutSstrains a single colony was inoculated into 200 ml
BMGY medium and grown as for Mut+ strains for 24 hours. The
culture was centrifuged and resuspended in 50 ml BMMY
medium containing 0.5% methanol, 0.5% glycerol and 1%
casamino acids. The cell suspension was divided into 2 shake
flasks, 25 ml in each, which were induced at 20uC and 30uC for
72 hours with daily addition of 1% of 1:1 mixture (v/v) of glycerol
During the cultivation 1 ml samples were withdrawn into
chilled 1.5 ml eppendof tubes and centrifuged at 16,0006g for
1 min. The supernatant and cell pellet were stored at 220uC until
To generate fermentation broth for purification larger volumes
were used as following. Single colonies of MutSstrains expressing
mutated version of phospholipase A1 were inoculated in 50 ml
BMGY medium in 500-ml volume shake flasks with baffles and
grown at 30uC for 16 hours with 150 rpm shaking. The whole
culture was used to inoculate 500 ml BMGY in a 2-L flask with
baffles and grown at the same conditions as before for 24 hours.
The cells were recovered by centrifugation, resuspended in 100 ml
BMMY medium with 0.5% methanol and 0.5% glycerol and
transferred to a 500-ml shake flask with baffles. The induction was
performed for 96 hours at 20uC with 150 rpm agitation and daily
addition of 1% 1:1 methanol:glycerol mixture. At the end of the
fermentation the biomass reached around ,130 g wet weight
biomass/L. The flasks were cooled on ice, the broth was
centrifuged at 8,0006g for 10 min at 4uC and decanted. 100 ml
of protease inhibitor cocktail (Sigma) was added and the broth was
stored at 220uC until purification.
Cloning and expression of antigen 5
The gene encoding antigen 5, Vesv5, was cloned from was
venom sac RNA into a TOPO vector (Invitrogen) . The gene
part encoding the mature peptide was amplified using primers
vesv5_fw and vesv5_rv (Table 1) and cloned into pPICZaA
plasmid as described above for Vesv1 gene. The resulting plasmid
pPICZaA_vesv5 was transformed into P. pastoris KM71H strain,
cultivated and the protein purified in the same manner as
described above for Ves v 1.
RNA isolation and RT-PCR
The yeast strains were cultivated as described above. 24 hours
after the start of induction the OD600of the cultures was measured
and the samples corresponding to 5?107cells (assuming OD600of 1
correspond to 107cells per ml) were quickly withdrawn into chilled
1.5 ml eppendorf tubes and briefly centrifuged at 16,0006g for
20 s. The supernatant was removed and the cell pellet resus-
pended in 200 ml RNALater solution (Ambion). The cells were
incubated on ice for 30 min, then briefly centrifuged, the
RNALater solution removed and the cells snap-frozen in liquid
nitrogen and stored at 280uC until RNA isolation. The total RNA
was isolated using RNAeasy Mini kit from Qiagen, the residual
DNA was removed using TURBO DNA-freeTMDNase from
Ambion. The RT-PCR was carried out using Titan One Tube
RT-PCR kit from Roche with AOX5 and AOX3 primers
(Table 1). To ensure that the product results from RNA and not
DNA, reactions with addition of only Taq polymerase instead of
reverse transcriptase and DNA polymerase mix were carried out in
parallel for all the reactions. The products of the reactions were
analyzed on 1% agarose gel strained with SYBR-SAFE (Invitro-
SDS-Polyacrylamide Gel Electrophoresis
The gel was a pre-cast 4–12% NuPAGEH NovexH Bis-Tris
electrophoresis gel from Invitrogen. The protein size marker was
unstained or pre-stained PageRulerTMprotein ladder from
Fermentas (Germany). The samples were mixed with NuPAGEH
LDS sample buffer (46) and NuPAGEH reducing agent (106) and
heated at 70uC for 10 min before loading on the gel. The
electrophoresis was performed at a constant voltage of 200 V for
35 min. The gel was used for western blot or stained with silver
stain (Fermentas, Germany).
Table 1. List of primers.
Primer Name Sequence Application
Amplification of vesv1 gene for cloning
into expression vector pPICZaA
vesv1_rv 59-GCACGTCTAGAGC GATGATTTTTCCTTTGTTGTTAC
vesv5_fw59- CATCCTCGAGAAAAGA AACAATTATTGTAAAATAAAATG
Amplification of vesv5 gene for cloning
into expression vector pPICZaA
vesv5_rv59- TACTTCTAGAGC CTTTGTTTGATAAAGTTC
Vesv1_S137A_fw 59-CAGATTGATCGGACACGCTTTGGGTGCTCACGCChange of serine137 to alanine
Vesv1_D165A_fw59-GAGATCATCGGATTGGCCCCTGCTAGACCTTCTChange of aspartate165 to alanine
Vesv1_H229A_fw59-CTTCTCCGAAGTTTGCTCTGCTAGTAGAGCCGTCATTTACChange of histidine229 to alanine
AOX3 59-GCAAATGGCATTCTGACATCColony PCR to test the integration of
inserts into the pPICZaA vector and RT-
The altered triplets are emphasized in the primers used for directed point mutagenesis of vesv1 gene. Recognition sites for restriction endonucleases XbaI (TCTAGA) and XhoI
(CTCGAG) are underlined.
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For western blot the proteins were elecrophoretically transferred
onto 0.2 mm PVDF membrane Amersham HybondTM-P (GE
Healthcare) using Mini Trans-BlotH electrophoretic transfer cell
(Bio-Rad Laboratories) at 100 V for 1 hour. The transfer was
done in 25 mM Tris, 192 mM glycine, and 20% v/v ethanol
buffer. The membrane was blocked with the blocking reagent
supplied together with Anti-His antibody (Qiagen) overnight,
incubated with Anti-His antibody in blocking buffer (1:1,000) for
1.5 hour, washed with TBST buffer for 10 min three times and
with TBS buffer for 5 min once. The bound antibody was detected
by chemoluminiscence with Amersham ECLTMAdvance Western
Blotting Detection Kit (GE Healthcare) and the signal was
recorded with camera [Hamamatsu Photonics, Japan].
Phospholipase A1 enzymatic assay was performed using
EnzChekH phospholipase A1 assay kit from Invitrogen according
to the manufacturer’s protocol with LecitaseH Ultra as a standard.
The fermentation broth from the cells transformed with empty
plasmids was used as background control. For the purified proteins
the storage buffer (PBS with 30% glycerol) was used as
background control. All measurements were performed at least
Purification with Ni-affinity chromatography
The proteins were recovered from the fermentation broth as
following. Glycerol was added to the broth to the final
concentration of 10% and 3 M NaCl to the final concentration
of 300 mM. Detergent Tween 20 was added to the final
concentration of 0.05%. The pH of the broth was adjusted to
7.5, the broth was centrifuged at 8,0006g for 15 min at 4uC and
filtered through 0.45 mm filter. A 1-ml column was packed with
Ni-NTA Superflow resin from Qiagen, washed with MilliQ water
for 3 column volumes (CV) and equilibrated with 20 CV of buffer
A (50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole,
10% glycerol, 0.05% Tween 20, pH 7.5) at 1 ml/min flow rate.
The broth was loaded on the column at 1 ml/min, the column was
Figure 1. AT-content of Vesv1 gene and predicted tertiary structure of Ves v 1 protein. Plotting of the AT-content of the Vesv1 gene was
performed using Gene Atlas utility (www.cbs.dtu.dk). The dark brown areas on the third line show DNA stretches with AT content above 80%. The 3D
structure of the Ves v 1 protein was generated using Geno3D (http://geno3d-pbil.ibcp.fr) using rat pancreatic lipase related protein 2 (1BU8) as a
template (29.4% identity, 43.7% similarity) . The structure was visualized in PyMOL 1.3 (http://www.pymol.org). The mutated amino acids are
shown in red.
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Figure 2. Analysis of fermentation samples on Western blot. 10 ml of fermentation broth after 3-day induction on methanol at 20 and 30uC
were analyzed by Western blot with anti-penta-His antibody. For several strains up to 4 transformants were tested and no significant difference
between clones was observed. The marker is a pre-stained PageRuler (Fermentas, Germany). Lot-to-lot variation of the apparent molecular weight of
pre-stained proteins in the ladder is ,5%.
Figure 3. Recombinant proteins concentration and enzymatic activity. Concentration of recombinant protein in fermentation broth (same
samples as on Figure 2) were measured by ELISA with anti-penta-His antibody (A). Phospholipase A1 enzymatic activity was measured in a
fluorometric commercial assay (Invitrogen) (B).
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washed with at least 20 CV of buffer A and the protein was eluted
with buffer B, which differed from buffer A by a higher imidazole
concentration of 250 mM. The fractions containing the protein
were pooled and desalted on ZebaTMDesalt Spin Columns
(Thermo Scientific). Protein concentration was measured by
amino acids analysis [24,25]. The proteins were resuspended in
PBS buffer with 30% glycerol and stored at 220uC.
Quantification of 6-HIS-tagged proteins by ELISA
Maxisorb microtiter plates (Nunc, Denmark) were coated with
100 ml samples diluted 1:10 or 1:100 in PBS buffer. The standard
curve was made with serial double-fold dilutions of purified rVes v
1 H229A, which concentration was determined by amino acids
analysis. The coating was performed at 4uC overnight. The plates
were washed with phosphate-buffered saline with 0.1% Tween20
(PBST) and blocked with 200 ml PBSTM (5% skim milk in PBST)
for 1 hour at room temperature. The plates were washed with
PBST and incubated for 2 hours at room temperature with anti-
penta-his HRP-conjugated antibody (Qiagen) diluted 1:500 in
PBSTM. The plates were washed with PBST after which 100 ml of
TMB ONE liquid substrate (Kem-En-Tek Diagnostics A/S,
Denmark) reagent was added to each well and the reaction was
allowed to proceed for 10 min at room temperature in the dark.
The reaction was terminated by addition of equal volume of 0.3 M
H2SO4. And the absorbance was measured at 450 nm using
Synergy reader (BioTek).
Quantification of IgE binding and inhibition by Enzyme
Allergo-Sorbent Test (EAST)
Maxisorb microtiter plates were coated with purified recombi-
nant allergens at the concentration 3 mg/ml in DPBS buffer
(Gibco) or with venom extract diluted with DPBS to the final
concentration of 10,000 SQ/ml. The coating was performed at
room temperature overnight. The plates were washed with ELISA
buffer (Pharmacy of the Danish Capital Region), blocked with
blocking/sample buffer (1% BSA, 0.1% Tween 20 in DPBS) for
2 hours at 37uC, washed again with ELISA buffer and incubated
with shaking at room temperature overnight with patients serum
diluted 1:10 in sample buffer. The plates were washed, incubated
with 1:250 Biotin Mouse a-Human IgE (BD) in sample buffer for
2 hours at 37uC, washed, incubated with 1:2000 ExtrAvidin-
Peroxidase (Sigma) in sample buffer for 30 min at room
temperature, washed again and developed with OPD reagent
(Dako) as recommended by manufacturer.
For inhibition assays the serum was pre-incubated with
recombinant allergen or wasp venom in sample buffer (1:20) at
4uC overnight before loading on the coated plates.
Histamine release assay
Peripheral blood mononuclear cells (PBMCs) from buffy coat
blood (non-allergic donor, anti-IgE responsive cells) were isolated
by Lymphoprep gradient centrifugation. IgE was stripped off the
basophils by a rebound in pH from 7.4 to 3.55 and back to 7.4.
The PBMCs were then incubated 1 hour with wasp patient or
control pool (non-allergic) serum to re-sensitize the basophils,
which subsequently were mixed with erythrocytes and challenged
in glass fiber coated microtiter plates (RefLab, Copenhagen,
Denmark) with wasp venom extract or with purified recombinant
allergens. Released histamine bound to the glass fibers was
coupled to o-phtahaldialdehyde, stabilized by HCLO4, and
measured fluorometrically as described previously . Results
were expressed as percentage of total cellular histamine content
and were considered positive when .10%.
Protein analysis by MALDI-TOF
Protein spots were picked from Coomassie-stained gels. Tryptic
digestion was carried out as previously described . The peptide
solution was applied on Anchorchip targetTM (Bruker Daltonics)
using CHCA as matrix. MS analysis was performed on a MALDI-
TOF-TOF Ultraflex II in positive ion reflector mode and spectra
were processed and analyzed using the software FlexAnalysis and
BioTools (Bruker Daltonics). Database searching was carried out
for each individual sample using an in-house MASCOT server
(Matrix Science, London, UK) to search NCBInr (ftp://ftp.ncbi.
nih.gov/blast/db/). The search criteria were selected as following:
peptide tolerance 650 ppm, fixed modifications – carbamido-
methyl (C), variable modifications – oxidation (M), allow up to 1
missed tryptin cleavages.
Cloning and mutation of phospholipase A1
We previously isolated a Vesv1 gene encoding wasp venom
phospholipase A1 from a local Danish species of V. vulgaris. 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). To avoid incomplete transcription of the
gene in the recombinant host we optimized the gene sequence
using online tool provided by Mr.Gene GmbH (Germany). During
the optimisation the most common P. pastoris codons were
preferentially used, though synonymous codons were used as well
to avoid excessive repetitions and restriction sites. We avoided
restriction sites necessary for cloning and plasmid linearization as
well as yeast splice donor sites, poly(A)-sites, TATA-boxes, RBS,
and 235 prokaria boxes.
Figure 4. Expression analysis of the active and mutated Vesv1
forms by RT-PCR. RT-PCR with primers binding to AOX1 promoter
and AOX1 terminator was performed on total RNA of wild type P.
pastoris and of P. pastoris expressing active and mutated versions of
Vesv1 gene, all the strains were induced on methanol for 24 hours. The
band of 2.2 kb corresponds to the native alcohol oxidase AOX1 gene,
which is expressed during growth on methanol. The 1.4 kb-band
corresponds to the recombinant Vesv1 transcript. In parallel to RT-PCR,
identical control reactions were carried out without addition of reverse
transcriptase. There were no bands detected in control reactions (not
shown) confirming the absence of DNA contamination.
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The synthetic gene was cloned in expression plasmid pPICZaA
after the strong methanol-inducible alcohol oxidase 1 (AOX1)
promoter and in-frame with the a-mating factor pre-propeptide for
tags at the C-terminus for easy detection and purification. The
resulting plasmid was mutated to substitute three different amino
acids from the predicted active site, serine137, aspartate165 and
histidine229 to alanine (Figure 1). The 3D modelling of the mutated
Ves v 1 variants was performed as well and the mutated versions
aligned well with the wild-type model, indicating that the introduced
mutations should not cause significant changes in the 3D structure of
the molecule. The resulting plasmids with wild-type and mutated
variants of the Vesv1 gene were introduced into two P. pastoris strains:
wildtype X33 strain with an active AOX1gene giving fastgrowth on
methanol (Mut+phenotype) and strain KM71H with a deletion in
AOX1 gene resulting in slow growth on methanol (MutSphenotype).
30uC. The induction of KM71H strain was performed with a 1:1
mixture of glycerol and methanol because we found in preliminary
experiments that it gave better final product titer than induction with
pure methanol (data not shown).
Expression of active phospholipase A1 and its mutated
Fermentation broth was analyzed for the presence and size of
recombinant proteins using Western blot (Figure 2). The
concentrations of recombinant proteins in the broth were
measured by ELISA and phospholipase A1 activity was analyzed
in enzymatic assay (Figure 3).
While phospholipase A1 enzymatic activity was detected in the
broth of cultures expressing a wild-type version of Vesv1 gene, the
protein could not be detected on Western blot under any
expression conditions. The reason could be too low expression
levels of the protein or partial degradation of the protein with a
loss of the C-terminal tag.
To ensure that Vesv1 gene was transcribed, we performed RT-
PCR (Figure 4) and could show that the gene was expressed,
though at a lower level than the mutated forms of Vesv1 if AOX1
expression is taken as a reference for comparison. The strains
expressing the active form of phospholipase A1 grew poorly on
methanol-containing plates in spite of the presence of the active
AOX1 gene as confirmed by colony PCR. We attempted high-cell
density cultivation of a P. pastoris strain expressing active Vesv1 gene
to see if higher yields could be obtained, however the culture went
into growth arrest at the switch to the inducing substrate methanol
even though the substrate was introduced at very low feed rate
(data not shown).
The mutated versions of rVes v 1 of the expected size of 36 kDa
could be detected in fermentation broth by Western blot. The
expression was slightly better at 20uC than at 30uC both in Mut+
and MutSstrains as measured by ELISA. The comparison
between the Mut+or MutSstrains is not very relevant in this
Figure 5. Purification of rVes v 1 H229A. The enzymatically inactive protein was purified from P. pastoris fermentation broth using Ni-affinity
chromatography. 10 ml samples of the fermentation broth, flow-through, wash and elution fractions were separated on SDS-PAGE and silver-stained.
The size marker is 5 ml of 10-fold diluted unstained PageRuler (Fermentas).
Wasp Venom Phospholipase A1 in P. pastoris
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contest as the optimal induction times for these two strains can
It can also be concluded that the proteins with modifications
S137A and D165A were in general expressed poorer than the
protein with modification H229A and this also correlates with the
presence of some residual phospholipase 1 activity in the
fermentation broth of those two variants, while H229A protein
does not have a detectable PLA1-activity. Silver-stained SDS-
PAGE of the fermentation broth of KM71H strains expressing
active rVes v 1 or partially active rVes v 1 D165A shows abundant
protein bands, while much fewer proteins and at lower
concentrations are seen for the strain expressing empty plasmid
or enzymatically inactive form of Ves v 1 (data not shown). This
could be a sign of cell lysis due to destruction of cell membrane by
the secreted phospholipase A1.
Purification and characterisation of enzymatically inactive
rVes v 1 H229A
We purified the rVes v 1 protein with mutation H229A from
fermentation broth of 120-hour shake flask culture of KM71H
strain. 1.7 mg/L was purified using Ni-affinity chromatography
(Figure 5). The purification conditions were quite stringent as we
wanted to obtain as pure protein as possible, otherwise higher yield
could be obtained. Besides the 36 kDa protein band correspond-
ing to rVes v 1 H229A monomer, a weak 72 kDa band was
observed in a few elution fractions with the highest protein
concentration. This is most likely a protein dimer appearing due to
inter-disulphide bridge formation. The 72-kDa artefact disap-
peared after protein desalting and dilution in storage buffer
(Figure 6). The 36 kDa protein was analyzed by MALDI-TOF
MS (Figure 6) and was identified as V. vulgaris phospholipase A1
Figure 6. SDS-PAGE gel of venom and purified rVes v 1 H229A
and rVes v 5. 1,000 SQ units of venom extract and 300 ng of purified
rVes v 1 H229A and rVes v 5 were separated on SDS-PAGE and stained
with coomassie. The identity of recombinant allergens and of the native
nVes v 1 and nVes v 5 proteins in the venom was confirmed by MALDI-
Figure 7. Binding of IgE antibodies from allergic patients sera to rVes v 1 H229A and rVes v 5 as measured by EAST. EAST results are
shown for twenty two sera. 5 sera (A, B, C, D and control serum) were chosen for further studies. Two sera (A and B) showed a positive response to
rVes v 1 H229A and negative to rVes v 5, one serum (C) showed the contrary, serum D was positive for both allergens, while control serum did not
react with either of the allergens.
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with 10 independent peptides matching with a mass tolerance of
50 ppm and with protein sequence coverage of 39% (p,0.05).
Several bands in the wasp venom extract were analyzed by
MALDI-TOF MS as well and the bands corresponding to the
native phospholipase A1 and antigen 5 were found with
significance p,0.05 (Figure 6). The recombinant proteins have a
size 2.7 kDa larger than the native allergens due to the presence of
the C-terminal tag.
IgE binding of rVes v 1 H229A
The ability of rVes v 1 H229A to bind specific IgE antibodies
was investigated in EAST assays with sera from the wasp-allergic
patients. The presence of antigen 5-specific IgEs was tested as well.
Twenty two sera were selected that had a high reactivity to wasp
venom and no reactivity to honey-bee venom in ImmunoCAP
tests. By choosing the sera that did not cross-react with honey-bee
venom we avoided the interference of specific IgE antibodies
directed towards carbohydrate determinants. Out of 22 sera 14
(64%) reacted with both rVes v 1 H229A and antigen 5, 4 (18%)
reacted with only Ves v 1 H229A, 2 (9%) with only antigen 5 and
2 (9%) did not react with either of the allergens (Figure 7). The
latter group of patients could be sensitized to other components of
the wasp venom as the peptide bone of hyaluronidase or dipeptidyl
peptidase IV. The control non-allergic sera did not react with
either of the recombinant allergens.
Two sera (A and B), which reacted with rVes v 1 H229A, but
not with rVes v 5, were chosen for inhibition study, where the
plates were coated with venom extract and the binding of sera to
the venom was inhibited with different concentrations of
recombinant rVes v 1 H229A (Figure 8). Binding of serum A to
wasp venom was completely inhibited both by venom itself and by
rVes v 1 H229A. For serum B the maximal inhibition by rVes v 1
H229A was 75%, whereas venom gave 96% inhibition.
Histamine release by rVes v 1 H229A
Histamine release (HR) from basophils sensitised with IgE
antibodies from allergic or control sera was used to test the
immunological activity of the recombinant allergens rVes v 1
H229A and rVes v 5, and wasp venom (Figure 9). The histamine
release assay is more sensitive than EAST inhibition and positive
response (above 10% histamine release) could be detected for
recombinant allergens concentrations as low as 10 ng/ml. As
illustrated in Figure 9, serum A showed histamine release with rVes
v 1 H229A and venom, but not with rVes v 5. Serum B, however,
showed release with both allergens, indicating that the lack of
detection of rVes v 5 response in EAST attributes to the lower
sensitivity of the assay.Reactivityof serum B IgE antibodies towards
rVes v5 explainswhythebindingtovenom could not becompletely
inhibitedwithrVes v 1 H229A in the EAST inhibitionassay.Serum
C, which was characterized as rVes v 1 H229A-negative, rVes v 5-
Figure 8. Inhibition of binding of two Ves v 1-allergic sera to wasp venom extract by rVes v 1 H229A. Microtiter plates were coated with
wasp venom and were incubated with sera mixed with different concentrations of either wasp venom or rVes v 1 H229A to test their ability to inhibit
the binding. Two sera A and B, which showed positive response to rVes v 1 H229A and negative to rVes v 5 in EAST assay, were chosen. The inhibition
percent shows the decrease of absorbance in comparison to the sample where no inhibitor was present. The shown inhibition values are averages of
two replicates. The absorbance A490values for non-inhibited samples were 1.1 for serum A and 0.9 for serum B.
Wasp Venom Phospholipase A1 in P. pastoris
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positive in the EAST assay, behaved in the same way in HR assay
mediating HR with only Ves v5 and venom. HR results for the Ves
v1 H229A and Ves v 5 double positiveserum (D) and for the control
serum were also consistent with EAST results, showing response for
both recombinant allergens and venom with serum D, but no HR
with control serum, respectively.
Specific immunotherapy (SIT) is used for treatment of various
allergies, where wasp and honey bee venom allergy, birch and
grass pollen allergy, house dust mites allergy are but a few
examples. Conventional allergy vaccines are partially purified
allergenic extracts, which can differ in batch-to-batch composition
and are difficult to standardize. The recent developments in
cloning and characterization of recombinant allergens have paved
the way for the new generation of recombinant allergy vaccines,
which can be produced with a high-consistent quality under good
manufacturing practice conditions. Moreover, they allow compos-
ing patient-tailored vaccines according to patients’ individual
sensitization profiles. Recombinant birch pollen allergen Bet v 1
and a cocktail of recombinant grass pollen allergens have been
Figure 9. Histamine release. Histamine release from human basophils sensitized with IgE antibodies from five sera (same as on Figure 7) was
tested when the basophiles were challenged with rVes v 1 H229A, rVes v 5 or wasp venom. The signal was considered positive when more than 10%
of the total histamine present in the basophils was released.
Wasp Venom Phospholipase A1 in P. pastoris
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shown to be efficient and safe for allergy treatment in human trials
[28,29]. To the best of our knowledge, there is no clinical data on
recombinant venom immunotherapy, though some animal testing
has been conducted showing efficacy of a honey bee fusion
vaccine, combining T-cell epitopes of three allergens , and of
wasp venom antigen 5 in murine model .
Availability of recombinant wasp venom allergens produced in a
safe expression host is an obstacle in developing recombinant
immunotherapy. While all of the allergens have been expressed in
baculovirus system and are well-suited for diagnostic purposes, for
therapeutic purpose it is desirable to produce the allergens in a
host well-suited for industrial production. One of the major
allergens from the wasp venom, antigen 5, was successfully
expressed in a eukaryotic host yeast P. pastoris previously, and here
we expressed another major allergen, phospholipase A1.
We previously found that expression of phospholipase A1 on the
surface of another yeast S. cerevisiae causes 70–82% growth
inhibition . Indeed our attempts to express an enzymatically
active phospholipase A1 in yeast P. pastoris resulted in defective cell
growth on plates and lead to cell lysis upon induction of expression
in fermentors. We therefore decided to express an enzymatically
inactive version of the protein instead. Such a protein, presuming
that its immunological and allergenic properties are preserved,
might be better suited for immunotherapy as it would not cause
side-effects due phospholipids degradation. We performed single
point mutations replacing the amino acids in the predicted active
site of the protein with alanine. The 3D modeling of the active and
mutated molecules did not show any significant changes of the
protein structure due to the mutations. Three mutated versions of
the allergen were expressed in P. pastoris. One of the variants, with
mutation H229A, had no detectable phospholipase A1 activity and
was secreted by P. pastoris at higher yields than other two forms.
Although enzymatically inactive, the protein preserved its IgE
binding activity as shown in EAST and inhibition EAST using
serum from wasp venom allergic patients, it was also immunolog-
ical active illustrated by its capability to mediated histamine release
from basophils sensitized with wasp venom specific IgE.
The yield of pure recombinant protein was 1.7 mg/L
fermentation broth. The yield can be further enhanced by strain
improvement, where either a rational approach can be undertaken
with optimization of promoter, signal sequence, copy number in
the genome or a simple screening of a larger number of clones can
be carried out. The fact that expression of Ves v 1 was higher at
lower temperature (20uC instead of 30uC) indicates possible
limited capacity of the cells to properly fold the given protein. If
this is the case, usage of a weaker promoter could be an advantage
as this would prevent the occurrence of the unfolded protein
response. Furthermore fermentation can be optimized, where high
cell-density cultivations can routinely give order of magnitude
higher yields than shake flask cultures.
In conclusion, we established expression of an enzymatically
inactive wasp venom allergen rVes v 1 in methylotrophic yeast P.
pastoris. The protein had histidine 229 in the enzyme active site
replaced by alanine. The protein showed immunological activity
in EAST and histamine release assay and could inhibit the binding
of Ves v 1-reactive sera to wasp venom. It presents a candidate of
recombinant immunotherapy, particularly in combination with
another major wasp venom allergen rVes v 5, which has also been
produced in P. pastoris.
We thank Anne Blicher from Enzyme and Protein Center (Technical
University of Denmark, Denmark) for amino acids analysis and for
assistance with MALDI-TOF MS analysis.
Conceived and designed the experiments: IB BMJ MAH LKP. Performed
the experiments: IB BMJ TW. Analyzed the data: IB BMJ LKP.
Contributed reagents/materials/analysis tools: IB BMJ MAH KLP. Wrote
the paper: IB BMJ MAH IS LKP.
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