Supplemental Material S1
Expression Constructs
PIMP
The PIMP open reading frame under the control of the AOX1 promoter and terminator
-factor signal sequence followed by a Kex2/Ste13 signal cleavage
site and the coding sequence of a fusion protein containing five different P. falciparum
(isolate 3D7) surface antigens: EGF-like domain of PfMSP4 (PlasmoDB Accession No.
PFB0310c, AA201-247), EGF-like domain 6 of PfRIPR (PlasmoDB Accession No.
PFC1045c, AA771-814), F2 part of the RII domain of PfEBA175 (PlasmoDB Accession No.
PF3D7_0731500, AA447-760 [T546A]), Pfs25 (PlasmoDB Accession No. PF3D7_1031000,
AA24-193 [T114A, T167A, S189A]) and the TSR domain of PfCSP (PlasmoDB Accession
No. PF3D7_0304600, AA311-383). As indicated in the square brackets four potential Nglycosylation sites (one within the RII domain of PfEBA175 sequence and three in the Pfs25
sequence) were removed by mutation of serine or threonine residues to alanine. The antigens
were connected by a three-alanine linker and a stop codon was added to the end of the coding
sequence. The resulting expression vector was named pPicZalphaA-PIMP.
PIMP_V1
A PCR product lacking the coding sequence for the four amino acid stretch EKRE in the
original PIMP sequence was generated by amplifying the N-terminal portion of the molecule
using primers PIMP_V1-Forward (5´TGC CAG CAT TGC TGC TAA AG´3) and PIMP_V1Reverse (5´GTC ATC CAA ATC GAT GTG AGC GGC CGC AAT ACA CTC AC´3) at the
same time introducing sequence overlaps compatible with pPicZalphaA-PIMP after digestion
with ClaI/XhoI.(New England Biolabs) The purified plasmid and PCR product were mixed in
equimolar amounts, incubated in assembly mixture (Gibson et al., 2009) at 50°C for 30 min
and then introduced into competent E. coli DH5alpha cells. The resulting expression plasmid
was named pPicZalphaA-PIMP_V1.
PIMP_V2
PIMP_V1 was used as a template to delete a 17-amino-acid stretch by conventional SOEPCR. The SOE product was generated in a two-step procedure using the primers PIMP_V2Forward (5´CCA TGG TCG AAT TCT TGG AGG ACG AGG´3) and PIMP_V2-SOEReverse (5´CAG TGT CAA CAG TAA CCG CGG CCG CTG GAC ACA TAG TAC AC´3)
as well as PIMP_V2-SOE-Forward (5´GGC CGC GGT TAC TGT TGA CAC´3) and
PIMP_V2-Reverse (5´GCT CTA GAT TAA GAA TTA ACA ACG´3) to generate two
overlapping PCR products in the first reaction, and the full-length PIMP_V2 variant using
PIMP_V2-Forward and PIMP_V2-Reverse. The SOE-PCR product was ligated to
pPicZalphaA-PIMP_V1 (first linearized with NcoI and XbaI) and the resulting expression
plasmid was named pPicZalphaA-PIMP_V2. All constructs were verified by sequencing.
Supplemental Material S2
Fed-batch fermentation
Inoculum preparation
For the first pre-culture, 3 mL YSG containing 100 µg ml-1 zeocin was inoculated with 30 µl
cell suspension from a selection plate and incubated overnight (28°C, 160 rpm). The second
pre-culture was prepared by transferring 3 ml of the first pre-culture into a 500-ml bottombaffled shake flask containing 200 ml YSG. The culture was then incubated for 24 h as above.
Fermentation
Cultivation was carried out in a BioBench 7 bioreactor (Applikon, Schiedam, The
Netherlands) with a working volume of 5 L and a H/D ratio of 1.5. The reactor was equipped
with three six-blade Rushton impellers, one L-sparger to ensure high gas flow rates and a
BlueInOne Ferm off-gas analyzer (BlueSens, Herten, Germany). Fed-batch cultivation was
initiated with 3 L reduced basal salts medium, containing 25 ml/L H3PO4 (85 %, v/v),
2.31 g/L MgSO4 ∙ 7 H2O, 0.18 g/L CaSO4 ∙ 2 H2O, 0.72 g/L KOH, 2.85 g/L K2SO4, 20 g/L
glycerol, and 0.25 ml/L Struktol J673 (Schill + Seilacher “Struktol” GmbH, Hamburg,
Germany) to prevent foaming. After sterilization and pH adjustment with 25% (w/w)
ammonia, 8 ml/L of Pichia trace metal (PTM) solution was added aseptically. The PTM
solution contained 0.10 g/L biotin, 0.01 g/L H3BO3, 0.10 g/L CoCl2 ∙ 6 H2O, 0.30 g/L
CuSO4 ∙ 5 H2O, 32.50 g/L FeSO4 ∙ 7 H2O, 2.50 ml/L H2SO4, 1.50 g/L MnSO4 ∙ H2O, 0.04 g/L
NaI, 0.10 g/L Na2MoO4 ∙ 2 H2O, 10 g/L ZnCl2. The cultivations were controlled under the
following conditions: temperature 25°C, aeration 1 vvm, and head pressure 1 bar. The pH was
maintained at 6.0 by the addition of 25% (w/w) ammonia. Except for the induction phase,
dissolved oxygen saturation (DO) was maintained above 30% by increasing the stirrer speed
from 500 to 1000 rpm. After inoculation with 100 ml of pre-culture all fermentations were
carried out according to a four-phase strategy: the first phase ended with the depletion of the
batch glycerol, indicated by a sharp rise of the DO value. Subsequently, the second phase was
initiated by feeding a 40% (w/w) glycerol solution at a constant flow of 14.9 g/hL initial
fermentation volume. The glycerol feed contained 0.048 ml PTM solution per gram glycerol.
Feeding was stopped after 16 h at an OD600 of ~290 and the methanol adaption phase (3rd
phase) was initiated by adding 0.25% (v/v) methanol. The fermentation volume was
calculated, taking all added solutions and removed sample volumes into account. After 3–4 h,
when a sharp rise in the DO value indicated the depletion of methanol, the adaption phase was
considered complete and methanol was fed, using a DO-based closed-loop control with a setpoint of 50% (4th phase). During this phase, the stirrer speed was set at 650 rpm. The
cultivation was stopped 24 h after the addition of the first volume of methanol by cooling the
fermentation broth to temperatures below 15°C. Afterwards the whole broth was harvested
and cells were separated from the supernatant by centrifugation in a Beckman Avanti J20
(Beckman Coulter, Brea, CA, USA) beaker centrifuge (9,000xg for 20 min at 4 °C). The
supernatant was collected and stored at -20 C in 450 mL-aliquots.
Analysis
Biomass concentration was measured as cell dry weight by centrifugation of 1.5 mL cell
culture at 16,000x g (BioFuge, Eppendorf, Hamburg) followed by the removal of the
supernatant. The pellet was dried to a constant weight in the oven at 80°C.
Supplemental Material S3
Mass spectrometry (MS)
For the protein identification, a protein fixed in the polyacrylamide gel was reduced, alkylated
and digested with trypsin (Promega, Mannheim, Germany) as previously described
(Shevchenko et al., 2006). The resulting peptides were analyzed by nanoHPLC (UltiMate
3000 HPLC system, LC PAcking, Dionex, Idstein, Germany) coupled to an amaZon ETD MS
ion trap spectrometer (Bruker Daltonics, Bremen, Germany) using ESI nano Sprayer. The
nanoHPLC system and the ion trap spectrometer were controlled using Bruker Compass
HyStar v3.2 – SR2 software. The liquid chromatography system was supplied with reversedphase pre-column (LC PAckings, Dionex) for sample desalting and a 15-cm PepMap 100
reversed-phase C18 column, 75 µm inner diameter (LC PAckings, Dionex) for peptide
fractionation. The peptides were separated using a 45 min linear gradient from 96% (v/v)
solution A (2% (v/v) acetonitrile, 0.1% (v/v) formic acid in high purity water) and 4% (v/v)
solution B (98% (v/v) acetonitrile, 0.1% (v/v) formic acid in high purity water) to 50% (v/v)
solution A and 50% (v/v) solution B at a flow rate 300 nl/min. The electrospray was operated
in positive ion mode with -4000 V spray voltage and 10 psi gas pressure. The end plate offset
of the mass spectrometer was set to -500 V and for the acquisition the standard method
Proteomics AutoMSMS Alternating Spectra CID-ETD Bruker trapControl v7.0 was used.
Raw data files were evaluated using Compass DataAnalysis v4.0 –SR5 software with
embedded search engine Mascot Search 2.3.01 (Matrix Science Ltd., London, UK). The
spectra were searched against the cRAP database 20100810 (144 sequences; 52207 residues)
including the PIMP sequence using the following parameters: enzyme trypsin, up to one
missed
cleavage
permitted,
no
fixed
modifications
and
variable
modifications
carbamidomethyl (C), oxidation (M) and propionamide (C) allowed, mass tolerance for
precursor ion ±0.3 Da and fragment ion ±0.3 Da.
Supplemental Figure S4
Figure S3. SDS-PAGE and immunoblot analysis of PIMP variants and quantification
by densitometry.
a) Coomassie stained gel. b) Immunoblot: Pfs25-specific murine monoclonal mAb 4B7 was
used to detect the PIMP variants. Visualization was performed using an alkaline
phosphatase-labelled goat α-mouse antiserum with nitroblue tetrazolium/5-bromo-4-chloro3-indolyl-phosphate solution as the substrate. c) Densitometric quantification of PIMP_V1
and PIMP_V2: The signal from the unidentified 72 kDa host cell protein was identified
(black peak area) and subtracted to calculate the yield of PIMP variants. 1: PageRulerTM
Prestained protein ladder, 2: Control (supernatant of a fermentation for the production of a
48 kDa P. falciparum antigen fusion containing Pfs25) 10 µL, 3: Supernatant from PIMP_V1
fermentation, 10 µL, 4: Supernatant from PIMP_V2 fermentation, 10 µL, 5: BSA standard
1500 ng, 6: BSA standard 1000 ng, 7: BSA standard 500 ng