Short report

Human Vaccines & Immunotherapeutics 9:11, 2342–2350; November 2013; © 2013 Landes Bioscience

Expression at a 20L scale and purification of

the extracellular domain of the Schistosoma
mansoni TSP-2 recombinant protein
A vaccine candidate for human intestinal schistosomiasis
Elena Curti1,2, Clifford Kwityn1,2,†, Bin Zhan1,2, Portia Gillespie1,2, Jill Brelsford3, Vehid Deumic3, Jordan Plieskatt3,
Wanderson C Rezende1,2, Eric Tsao4, Bose Kalampanayil4, Peter J Hotez1,2,5, and Maria Elena Bottazzi1,2,*
1
Departments of Pediatrics and Molecular Virology and Microbiology; National School of Tropical Medicine; Baylor College of Medicine; Houston, TX USA; 2Sabin
Vaccine Institute and Texas Children’s Hospital Center for Vaccine Development; Houston, TX USA; 3Department of Microbiology; Immunology and Tropical Medicine;
the George Washington University; Washington, DC USA; 4Aeras; Rockville, MD USA; 5James A. Baker III Institute for Public Policy; Rice University; Houston, TX USA

†
Current affiliation: Beckman Coulter Inc.; Chaska, MN USA

Keywords: Schistosomiasis, Schistosoma mansoni, neglected tropical dis-

eases, vaccine, Sm-TSP-2, Pichia Pastoris, recombinant protein

A novel recombinant protein vaccine for human schistosomiasis caused by Schistosoma mansoni is under develop-
ment. The Sm-TSP-2 schistosomiasis vaccine is comprised of a 9 kDa recombinant protein corresponding to the extracel-
lular domain of a unique S. mansoni tetraspanin. Here, we describe the cloning and the expression of the external loop of
Sm-TSP-2 recombinant protein secreted by Pichia Pink™ the process development at 20L scale fermentation, and the two-
steps purification, which resulted in a protein recovery yield of 31% and a protein purity of 97%. The developed processes
are suitable for the production of purified protein for subsequent formulation and Phase 1 clinical studies.

Introduction
Schistosomiasis is a parasitic infection caused by blood flukes
of the genus Schistosoma. Today, human schistosomiasis is con-
sidered one of the most important human helminthic infections
in terms of mortality and morbidity, especially in Africa, where
more than 90% of the cases occur.1,2 Recent studies indicate that
approximately 200 million people are infected worldwide, with
800 million people at risk.3-6 However, additional analyses indi-
cate that this number may be an underestimate and as many as
400–600 million people may be infected with schistosomes.7
Although schistosomiasis is a treatable infection, the current
treatment of choice does not provide an optimal strategy for
controlling the disease.8 Since 1990, praziquantel (PZQ) based
mass chemotherapy has been the major approach in controlling
schistosomiasis, by primarily targeting school-aged children with
annual treatments.9 However, the sustainability of PZQ treat-
ment for the long-term control and elimination of schistosomi-
asis remains a concern and has limitations. For instance, PZQ
does not prevent reinfection nor does it reduce transmission in
highly endemic areas of Africa and elsewhere.10-12 In addition,
there is an increasing concern about the development of parasite
resistance to PZQ. Although there is no clear evidence for the
existence of PZQ-resistant schistosome strains, decreased suscep-
tibility to the drug has been observed in several countries.13-15 To
overcome these challenges, a prophylactic vaccine or a vaccine-
linked chemotherapy would be ideal to complement the existing
treatment initiatives.16 Evidence for the feasibility of developing a
schistosomiasis vaccine includes studies showing that immuniza-
tion with irradiated schistosome cercariae regularly induces high
levels of protection in experimental animal models, with boosts
further increasing the level of protection.17 In addition, a subset
of human populations living in endemic areas has been shown to
develop various degrees of natural resistance,18 while veterinary
recombinant vaccines against other platyhelminth parasites have
been developed successfully and applied in practice.19
Expanded information on the mechanisms of immunity
to schistosomiasis20 and the recent availability of the schisto-
some genome for both S. mansoni and S. hematobium21 have
resulted in the discovery of several schistosome antigens, while
additional candidates are now being found through proteomic
approaches.22-24 Within the last year results of a Phase 1 trial for
a Sh28GST (recombinant 28 kDa glutathione S-transferase)
vaccine against S. hematobium, the cause of urogenital

*Correspondence to: Maria Elena Bottazzi; Email:bottazzi@bcm.edu

Submitted: 04/02/2013; Revised: 07/02/2013; Accepted: 07/17/2013
http://dx.doi.org/10.4161/hv.25787

2342 Human Vaccines & Immunotherapeutics Volume 9 Issue 11

 Short report Short report

Figure 1. (A) Coomassie stained SDS-PAGE of 10 µl culture of recombinant Sm-TSP-2 expressed in PichiaPink™ strain #4 after induction with 0.5%
methanol for 72 h; (B) Coomassie stained SDS-PAGE and Immunoblot of recombinant Sm-TSP-2 expressed in PichiaPink ™ strain #4 after induction with
0.5% methanol for 72 h, blot was probed with rabbit anti-Sm-TSP-2 serum. The volume of culture loaded in each lane is reported above each lane and
expressed in µl. (C) Coomassie stained SDS-PAGE of recombinant Sm-TSP-2 expressed in PichiaPink™ strain #4 in BMMY medium with different pHs. WB
indicates without buffer. SB indicates SeeBlue® Plus2 Pre-Stained Standard. The full line arrow indicates the full length protein; the dotted line arrow
indicates the clipped form.

schistosomiasis, were reported.25 Moreover, the Oswaldo Cruz
Foundation (FIOCRUZ) recently announced the start of a Phase
1 trial for a S. mansoni vaccine using a recombinant fatty acid
binding protein known as Sm14 (http://apps.who.int/trialsearch/
trial.aspx?trialid=NCT01154049).
Several new antigens are now being discovered through a
combination of reverse vaccinology and OMICs approaches.26,27
Among them are the tetraspanins, which represent a group of
highly abundant proteins in the tegument, the outermost mem-
brane of the intra-mammalian stage of the parasite, and there-
fore exposed and accessible to the host immune system.23,24
Tetraspanins are characterized by four transmembrane domains,
which are linked by two extracellular loops, a short loop of about
20 amino acids, and a longer loop of about 70–90 amino acids.27
Table 1. Stages of the Sm-TSP-2 upstream process
While the shorter loop has little tertiary structure, the longer
loop contains four to six cysteines that form disulfide bonds.
Genomics, combined with two signal sequence trapping stud-
ies, were used to identify and clone two S. mansoni tetraspanin,
TSP-1 and TSP-2.26 The full length Sm-TSP-2 protein is thought
to have a role in both tegument formation and maturation as the
ultra-structural morphology of the adult worm. Scistosomulae
treated in vitro with Sm-TSP-2 double strand RNA (RNAi)
displays a distinct vacuolated and thinner tegument compared
with the untreated controls.28 The full length Sm-TSP-2 encom-
passes 239 amino acids of which residue 107 to 184 make the 78
residues long extracellular domains. The long extracellular loop
of Sm-TSP-2 has proven to be an effective anti-schistosomiasis
vaccine, eliciting 57–64% protection in mice vaccinated with

Inoculum Culture
Step Inoculum Growth conditions
volume volume
Preparation of
Seed stock 3 mL 15 mL N/A
seed stock
Preparation of 2L flasks Seed stock 1.6 mL 0.8 L 28.0 ± 1 °C, 180 ± 10 rpm, 21 ± 3 h, BMG medium
Inoculation of the
2L flasks 1.6L 20 L 9L BMG medium pH 5.0, 28 ± 1 °C, 30% O2, 457 ± 10 rpm, 0.33 ± 0.1 bar, 17 ± 2 hours
20L Fermentor
Feed batch glyc- 50% (v/v) glycerol flow rate of 15 g/L/h, for 6 h, 457 RPM (pH was adjusted from
N/A N/A 20 L
erol phase 5.0 to 4.5 and temperature adjusted from 28.0 °C to 25.0 °C at the fifth hour)
20 L
Methanol ramp feed N/A N/A Flow rate from 1.2 mL/L/hr to 8.7 mL/L/h for 8 h, 640 RPM, pH 4.5, 25 ± 1 °C

Methanol induction N/A N/A 20 L 100% methanol at a flow rate of 11 ml/L/h for 57 h, 640 RPM, pH 4.5, 25 ± 1 °C
The table describes the stages of the upstream process, the inoculum with respective volume, the culture volume and growth conditions for each step are
described.

www.landesbioscience.com Human Vaccines & Immunotherapeutics 2343

the antigen followed by challenge with S. man-
soni cercariae.26,27 Moreover, the recombinant
extracellular domain of the Sm-TSP-2 protein
is also strongly recognized by IgG1 and IgG3
from putatively resistant, but not from chroni-
cally infected individuals further highlighting
the promise of this antigen as a subunit vaccine
against human schistosomiasis.29
Identification of new antigens and successful
protective results are of little value if recombinant
proteins cannot be produced easily (and cheaply)
with good manufacturing practices (GMP) and
in large scale. Here we describe the cloning of the
large extracellular loop of Sm-TSP-2, its expres-
sion in the PichiaPink™ yeast expression host
and the development of a process, which is con- Figure 2. Coomassie stained SDS PAGE under reducing (A) and non-reducing conditions (B),
sistent, reproducible and allows producing stable of the Sm-TSP-2 expression. In both A, and B: lane 1: Mark12™ Unstained Standards, lane 2:
pre-methanol induction; lane 3: 24 h post-methanol induction; lane 4: 48 h post-methanol
recombinant protein that can be manufactured induction; lane 5: 65 h post-methanol induction; lane 6: harvest material.
and serve as a subunit vaccine candidate antigen.
Results
Construction of the recombinant expression plasmid
The DNA fragment optimized for expression in PichiaPink™
was inserted between the XhoI and SwaI site of pPinkα-HC, which
contains the alcohol oxidase AOX1 promoter and allows for metha-
nol induced expression. Sequencing of the recombinant plasmid
confirmed that the reading frame of Sm-TSP-2 was correct.
Transformation, small scale expression and generation of
seed stock
The recombinant DNA was transformed into the protease A
and B deficient PichiaPink™ strain #4 by electroporation, and
transformants were selected with a Pichia Adenine Dropout
(PAD) agar plate. Twelve transformants were induced at a
small scale with 0.5% methanol and the clone with the highest
expression level was selected for making a research seed stock in
yeast peptone dextrose (YPD) liquid media with 20% glycerol.
After being stored for 6 months at −80 °C, the research seed stock
of Sm-TSP-2/pPinkα-HC/PichiaPink™ #4 had high viability
(2.15 × 108 CFU/ml) and had ability to express the Sm-TSP-2
extracellular domain recombinant protein.
The induction conditions were optimized to ensure maximal
levels of expression, while minimizing the degradation of the
expressed Sm-TSP-2 extracellular domain recombinant protein.
SDS-PAGE of the fermentation supernatant revealed a major pro-
tein migrating at an apparent molecular weight of 9 kDa indicat-
ing that the extracellular domain of the Sm-TSP-2 recombinant
protein, which has a predicted molecular weight of 8813.06 Da
and a pI of 8.92, was successfully expressed in the PichiaPink™
host and secreted in the supernatant (Fig. 1A and B). Expression
was confirmed by immunoblot using rabbit Sm-TSP-2 specific
antibodies (Fig. 1B). To optimize the protein expression yield,
various fermentation media, and different pHs were investigated.
The induction conditions that resulted in minimal degrada-
tion and maximum yield were achieved in buffered methanol-
complex medium (BMMY) at pH 3.0–5.0 for 72 h (Fig. 1C).
2344 Human Vaccines & Immunotherapeutics
However, even using the optimal fermentation conditions, both
SDS-PAGE analysis and immunoblot of the cell free supernatant
samples harvested at different fermentation times revealed that a
smaller protein, with a lower apparent molecular weight, was also
secreted in the supernatant along with the 9kDa recombinant
fragment (Fig. 1C; Fig. S1). Full sequencing of the 9 kDa protein
and of the lower molecular weight product showed the smaller
specie to be a hydrolytically cleaved clipped form of the final puri-
fied material (Fig. 1A and B). Thus, the Sm-TSP-2 extracellular
domain recombinant protein experienced proteolysis through the
actions of both specific yeast proteases and exopeptidases during
expression (Fig. S1). Several sites in the molecule were identi-
fied as vulnerable to the action of these hydrolases. Three KK
sites and one RK site, that are vulnerable for a host yeast Kex-2
protease, as well as the two C-terminal amino acids susceptible
to carboxypeptidase cleavage were determined and confirmed by
both mass spectrometry and N-terminal sequencing (Fig. S1).
Scale up expression of the Sm-TSP-2 extracellular domain
recombinant protein
The expression of the extracellular domain of Sm-TSP-2
was scaled up to 20L fermentation in BMG as summarized in
Table 1. Cells were grown at 28 ± 1 °C in 30% dissolved oxygen
with an agitation speed of 457 rpm and pressure of 0.33 ± 0.1 bar.
Approximately 17 ± 2 h after inoculation and a sharp increase in
the percentage of dissolved oxygen pO2, 50% (v/v) glycerol was
introduced into the cell culture medium for 7 h. Shortly after
the glycerol batch phase, the methanol induction phase was initi-
ated. In order not to overdose the fermentation with methanol,
a DO spike was allowed before initiating the methanol ramp
feed. Methanol induction was continued for another 57 h, for
a total of 65 h, by addition of 100% methanol until harvest. In
the scaled up fermentation a 65 h induction period was preferred
to the 72 h to minimize cost of production. A distinct band of
approximately 9kDa was observed after 24 h induction, whereas
there was no expression of Sm-TSP-2 recombinant protein before
induction (Fig. 2). The Sm-TSP-2 recombinant protein secreted
Volume 9 Issue 11
Table 2. Stages of the Sm-TSP-2 downstream process

Stage Steps Description

The starting material, microfiltration supernatant (permeate), is concentrated about 10-fold and buffer
Stage 1 UF/DF
exchanged into the SP Sepharose XL column loading buffer.
The buffer exchanged supernatant is loaded onto the SP Sepharose XL column. Sm-TSP-2 binds to the col-
umn while the majority of host cell proteins, DNA and other impurities are collected in the column flow-
Stage 2 SP Sepharose XL
through. After washing the SP Sepharose XL column with Succinic acid buffer pH 6 (~15 mS/cm) the bound
Sm-TSP-2 is eluted with a linear gradient using 500 mM sodium chloride.
The eluted Sm-TSP-2 from the SP Sepharose XL column is concentrated 2-fold and buffer exchanged into
Stage 3 UF/DF
the formulation buffer prior to the Q Sepharose HP column.
The buffer exchanged Sm-TSP-2 is applied to a Q Sepharose HP equilibrated with formulation buffer. The
Stage 4 Q Sepharose HP remaining host cell protein and impurities bind to the Q Sepharose HP column, while the non-binding TSP-2
is collected in the column flow-through.
0.22 µm Filtration/ The collected Sm-TSP-2 fraction from the Q Sepharose HP column is filtered through a 0.22 μm filter in a Bio
Stage 5
Storage Safety Cabinet. The final Sm-TSP-2 Bulk is stored at -80 °C.
The table describes the stages of the downstream process, the steps included, and the description of each step.

in the fermentation supernatant accounted for 81% of the total
protein in the medium with a yield of approximately 8 g as quan-
tified by SDS-PAGE followed by densitometry.
Recovery and purification of the Sm-TSP-2 extracellular
domain recombinant protein
The Sm-TSP-2 extracellular domain recombinant protein was
purified with a two-step chromatographic process which is sum-
marized in Table 2. Following purification the overall yield of
protein recovery was 31% with 97% purity. The mass balance of
the process is described in Table 3. Quantification of the protein
in the downstream process samples was performed by RP-HPLC
and SDS-PAGE. Figure 3 shows representative elution profiles of
the in-process samples harvested at the different steps of the down-
stream process. In all samples analyzed, the protein eluted from
RP-HPLC column as a major peak with an average retention time
of 9.8 min. The same in-process samples were also analyzed by
SDS-PAGE followed by Coomassie Blue staining (Fig. 4A and B).
SDS-PAGE followed by Coomassie Blue stain was used as a sec-
ond orthogonal method to evaluate the purity and identity of the
samples. Analysis of the eluate following passage through the SP
Sepharose XL resin (Fig. 3B) shows the removal of both high and
low molecular weight contaminants during this caption step. After
the SP Sepharose XL column the Sm-TSP-2 recombinant protein
was 96.7% pure and the clipped Sm-TSP-2 protein was removed
(Figs. 3B and 4A–B). The remaining host cell proteins were
removed during the Q Sepharose HP step, with only a nominal
loss of the target protein. Analysis of the elution sample from the Q
Sepharose HP column indicated that the protein was 97.4% pure.
Identity of the purified protein was confirmed by mass spectrom-
etry (Fig. S3), immunoblot analysis (Fig. S2) as well as N-terminal
sequencing (Fig. S1) and AAA analysis (Table S1). In addition to
the main peak of 8809 Da, the mass spectrometry profile of the

Table 3. Mass balance and purity of Sm-TSP-S 2 as determined by RP-HPLC

Sm-TSP-2 Sm-TSP-2 Recovery

Process Steps Purity (%)
amount (g) (% of the starting material)
Not induced 0 ND ND
24 h induction 5.3 ND ND
48 h induction 7.99 ND ND
Upstream
65 h induction 8.6 ND ND
Harvested supernatant 8.61 100 81
0.2 µm filtered supernatant* 6.80 79 81
UF/DF retentate* 5.52 64 81
SP Sepharose XL load* 5.6 64 81
Elution 2.65 33 96.7
Downstream UF/DF retentate 2 2.80 32.5 97.0
Q Sepharose HP load 2.70 31.3 97.2
Q Sepharose HP flow through 2.71 31 97.4
Purified protein 2.8 31 97
Upstream and downstream in process samples were analyzed by RP-HPLC, except for those samples denoted by an * which were analyzed by SDS-PAGE
(ND, not determined).

www.landesbioscience.com Human Vaccines & Immunotherapeutics 2345

purified sample showed an additional peak with a
molecular weight of 8907 (Fig. S3). The 98 Da rela-
tive differences between this peak and the main peak
were consistent with post-translational phosphoryla-
tion. The other three minor peaks, accounting for
5%, of the total species observed differed from the
parental peak by multiple of 162 Da suggesting that
approximately 5% of the Sm-TSP-2 recombinant
protein underwent up to three glycosylation events,
likely N-mannosylation.

Discussion

The successful development of a vaccine to

combat human schistosomiasis has proven elu-
sive to date. While irradiated live whole organism
schistosome vaccines induce a high level of pro-
tective immunity in laboratory animals, the first
generation of recombinant protein and DNA vac-
cines (obtained through immunoscreening with
sera from chronically infected individuals) failed
to show a comparable or reproducible level of pro-
tection. To overcome this problem a new approach
that combined genomics, and high throughput
proteomics of the worm’s outer syncytial surface
(also known as the tegument) with targeted host
immunity from putatively resistant individuals has
recently yield an exciting and previously undiscov-
ered family of candidate antigens which belong to Figure 3. RP-HPLC elution profile of in process samples from (A) SP XL load; (B) SP XL
the family of tetraspanins. 26,29
In particular, signal elution, (C) Q HP flow through.
sequence trapping studies were used to capture
two S. mansoni tetraspanin, TSP-1 and TSP-2. In
extensive preclinical testing, the extracellular loop domain of The process yielded approximately 2.5 g of the recombinant pro-
tetraspanin-2, Sm-TSP-2, has consistently conferred high levels tein, representing approximately 31% recovery, of the extracel-
of protection in several S. mansoni challenge models.26,30 Equally lular excreted recombinant protein assessed by RP-HPLC and
compelling is that Sm-TSP-2 is uniquely recognized by sera from SDS-PAGE to be more than 97% pure. A detailed analysis of in-
individuals resistant to S. mansoni infection in Brazil.27 These process samples, directed to assess the protein content and purity
studies led to the selection and process development of the extra- at each step showed that protein loss occurred predominantly in
cellular domain of Sm-TSP-2 recombinant protein as a subunit two of the process steps. A 50% loss was observed during the
vaccine candidate for Phase 1 trials in humans. initial microfiltration and 0.22 µm filtration prior to concentrat-
An ideal schistosomiasis vaccine would consist of a single- ing using the UF/DF system and a 20% loss observed during the
dose of a highly purified and stable formulation, which would be SP Sepharose XL chromatographic step. The process was highly
administered following praziquantel treatment. However, devel- effective in eliminating both high molecular weight host derived
oping a recombinant protein production process suitable for GMP contaminants, lower molecular weight product derived species,
manufacturing at an industrial scale is complex and challenging and the clipped form of Sm-TSP-2 extracellular domain recom-
as it requires the ability to manufacture and scale up production binant protein resulting from proteases activity. Preliminary
and ensure stability of the candidate antigen throughout its shelf stability analysis had in fact determined that the Sm-TSP-2 extra-
life. In addition, as schistosomiasis affects predominantly the cellular domain protein experiences hydrolytic cleavage through
world’s poorest people, the necessity to manufacture a vaccine at the actions of both specific yeast proteases and exopeptidases
lowest cost possible adds to the challenge. To this end, bacterial while being expressed and secreted in the fermentation super-
(Escherichia coli) and yeast (Pichia pastoris) expression systems natant. Both specific yeast proteases and exopeptidases activities
were prioritized and explored. However, because of the potential were halted by the process described. Mass spectrometry analy-
for contamination of pyrogenic components derived from E. coli sis confirmed that the recombinant protein exhibited a mass of
and other issues related to solubility and scale-up. PichiaPink™ 8907, which closely matches the predicted molecular weight of
was chosen as the most suitable expression host to produce large the molecule. Less than 5% of the overall total species under-
amounts of Sm-TSP-2 recombinant protein for clinical studies. went to phosphorylation or glycosylation events. In addition, the

2346 Human Vaccines & Immunotherapeutics Volume 9 Issue 11

 of the culture. Once the “OD PROXY”
culture reached an OD600 nm ≥ 3.5,
monitoring of the other two shake flask
cultures OD600 nm was started until
the target OD600 nm of 9.0 ± 0.2 was
reached. The flasks were then harvested
and used to inoculate a 20 L fermen-
ter containing 9 L of basal salt media
(BSM). The fermentation was con-
ducted in a 20 L Bioengineering fermen-
ter, model NLF-22 (Bioengineering).
The pH of the BSM was adjusted prior
Figure 4. Coomassie stained SDS PAGE under reducing (A) and non-reducing conditions (B) of the to inoculation to 5.00 ± 0.2 and the pH
upstream and the downstream in process samples. A and B: lane 1: Mark12™ Unstained Standards; maintained at this set point with addi-
lane 2: Cell free supernatant; lane 3: UF/DF retentate prior to SP XL; lane 4: SP XL elution; lane 5: UF/
tion of 14% ammonium hydroxide.
DF retentate prior to Q HP; lane 6: Q HP flow through.
Cells were grown at 28 ± 1 °C with a
dissolved oxygen set point of 30% and
purification technique is non-denaturing, hence is not expected at an agitation speed of 457 rpm and pressure of 0.33 ± 0.1
to affect the immunogenicity of the protein as documented in the bar. Approximately 17 ± 2 h after inoculation and after a sharp
cited references. Undergoing stability studies performed under increase in the percentage of dissolved oxygen pO2 (indicating
a quality control programs have also confirmed stability of the depletion of glycerol), 50% (v/v) glycerol was introduced into
Sm-TSP-2 recombinant protein for one year post manufacturing. the cell culture medium at a set flow rate of 15 g/L/h, for 7 h.
In conclusion, we have developed a novel, highly efficient and Five hours after the glycerol fed batch was initiated, the pH
cost effective process that indeed provides a proof of principle for was ramped from 5.0 to 4.5 and the temperature adjusted from
a scalable process and its suitability for a Phase I trial in humans. 28.0 °C to 25.0 °C for a two hour period prior to the comple-
Ultimately, further process improvements may be required tion of glycerol fed batch phase. After the seven hours glycerol
to increase the yields, improve process recovery and therefore fed batch phase, the agitation was increased from 457 to 640
reduce the costs of manufacturing and advance the production RPM. Excessive foaming was controlled with addition of 10%
for downstream Phase II and III trials. (v/v) antifoam KFO673 (KABO Chemicals Inc.) in deionized
water. Shortly after the glycerol fed batch was stopped another
Materials and Methods pO2 spike was observed. The methanol ramp was initiated at a
flow rate of 1.2 mL/L/hr and over 8 h ramped to 8.7 mL/L/hr.
Cloning and generation of research seed stock A decrease in the pO2 in the fermentation media was observed
The DNA sequence of the extracellular domain of Sm-TSP-2 after addition of methanol. In order not to overdose the fermen-
was codon optimized for expression in PichiaPink™ Yeast tation with methanol, a DO spike was allowed before initiating
Expression System (Invitrogen) and cloned into the expression the methanol ramp feed. Methanol induction was continued
vector pPinkα-HC using XhoI and SwaI sites. PichiaPink™ for another 57 h (for a total of 65 h) by adding 100% methanol
Strain 4 (ade2, prb1, pep4) transformants were induced at small at a flow rate of 11 ml/L/h until harvest. The total amount of
scale with 0.5% methanol and the clone with the highest expres- methanol used during the induction period was 5081 mL.
sion level was selected for making a seed stock in Yeast Peptone Downstream manufacturing processes
Dextrose (YPD) liquid media with 20% glycerol. The seed stock Clarification and ultrafiltration/diafiltration (UF/DF)
was stored frozen at −80 °C. After 65 h of methanol induction, the fermentation was com-
Upstream manufacturing process pleted and cells were removed by microfiltration using a 750 000
Expression of Sm-TSP-2 mwco filter (Millipore) and five diafiltration volumes of 50 mM
The fermentation process as summarized in Table 1 was ini- succinic acid buffer with 30 mM NaCl pH 4.6. Following the
tiated with the expansion of the seed stock into shake flasks clarification, the filtrate was passed through a 0.22 µm filter. The
containing 800mL of buffered medium with glycerol (BMG) to clarified fermentation supernatant was then filter sterilized, and
use as an inoculum for the fermentation. Briefly, approximately concentrated to a volume of approximately 8 L before being dia-
3 mL of the seed stock were transferred into one sterile 15 mL lyzed using a UF/DF plate filter with a surface area of 2m2 and a
conical tube to make a seed culture. Approximately 1.6 mL of molecular weight cut off of 3 kDa. The Sm-TSP-2 recombinant
seed culture was then used to inoculate each of the three 2 L protein was dialyzed in 50 mM succinic acid buffer with 30 mM
shake flasks, containing 800 mL of BMG medium. The flasks NaCl, pH 4.6 (SP Sepharose XL load condition) until a pH of
were incubated at 28.0 ± 1 °C with agitation of 180 ± 10 rpm, the permeate reached 4.6 ± 0.2 and the conductivity was between
for approximately 21 ± 3 h, before monitoring the cell density the range of 6.5 ± 1.3 mS/cm. Prior to loading on the column
(OD600 nm). Initially, one of the three flasks was used as an the UF/DF retentate was filtered through a 0.22 µm Millipak
optical density proxy to monitor the increase in OD600 nm 60 filter.

www.landesbioscience.com Human Vaccines & Immunotherapeutics 2347

 SP Sepharose XL column chromatography
The Sm-TSP-2 extracellular domain recombinant protein
was bound to a SP Sepharose XL column (cation exchange)
connected to an Akta Pilot system (GE Healthcare). The SP
Sepharose XL column (7 × 13.2 cm, GE Healthcare) was prepared
according to the manufacturer’s instructions, sanitized with 0.5
N NaOH, and equilibrated with a 50 mM succinic acid buffer
with 30 mM NaCl buffer at pH 4.6. All steps were performed at
room temperature at a flow rate of 150 cm/h (96 mL/min). After
adjustment of the pH and conductivity to 4.6 ± 0.2 and 6.5 ± 1.3
mS/cm respectively, the harvest material was loaded onto the SP
Sepharose XL column and washed with 2–3 column volumes of
50 mM succinic acid buffer with 30 mM NaCl pH 4.6, followed
by 4–5 column volumes (CV) with 50 mM succinic acid buffer
with 85 mM NaCl, pH 6.0 buffer to remove host cell protein.
The protein was eluted with a 12 CV linear gradient (85 mM
NaCl–500 mM NaCl, 10%B-65% B). Elution of the Sm-TSP-2
protein was monitored by A280 using the AKTA Pilot system
controller screen. The gradient produced three distinct peaks.
The third peak corresponding to the Sm-TSP-2 protein was
collected and evaluated by SDS-PAGE and Reverse Phase High
Performance Liquid Chromatography (RP-HPLC).
Diafiltration and ultrafiltration
The Sm-TSP-2 eluted from the SP Sepharose XL column was
diafiltered into 10 mM imidazole, 15% sucrose, 2 mM phos-
phate, pH 7.4 by ultrafiltration using a 3 kDa membrane with 1
m2 surface area (Millipore). This was installed into the cleaned
Millipore Pellicon UF system. The UF/DF system was primed
and equilibrated with 10 mM imidazole, 15% sucrose, 2 mM
phosphate pH 7.4 until the pH value for the permeate was 7.4
± 0.2 and the conductivity 0.6 ± 0.2 mS/cm. The concentrated
SP Sepharose XL pool was diafiltered with at least 8 CV of 10
mM imidazole, 15% sucrose, and 2 mM phosphate pH 7.4. Prior
to loading on the next chromatography column, the Sm-TSP-2
was filtered through a 0.2 µm Millipak 60 filter. The diafiltered
Sm-TSP-2 recombinant protein was further purified using a neg-
ative capture anion exchange Q Sepharose HP column.
Q Sepharose HP column
The Q Sepharose HP (5.0 × 11 cm, GE Healthcare) was used
as a negative capture (non-binding) second chromatographic
step. The Q Sepharose HP column was prepared according to
the manufacturer’s instructions. After sanitization, the column
was equilibrated with 3 CV’s of 10 mM imidazole, 15% sucrose,
2 mM phosphate pH 7.4 at 49 mL/min (150 cm/hr). The equili-
bration of the Q HP column was completed when the column
effluent pH was 7.4 ± 0.2 and conductivity was 0.6 ± 0.2 mS/cm.
The diafiltered Sm-TSP-2 recombinant protein was loaded onto
the Q HP column at temperature of 15–25 °C and a flow rate of
~150 cm/h (49 ml/min). The Sm-TSP-2 protein eluted from the
column in the flow-through as monitored by A280. The eluate
was then filtered through a 0.22 μm filter and stored at −80 °C.
Characterization of recombinant Sm-TSP-2 extracellular
domain
SDS-PAGE
Analysis of Sm-TSP-2 extracellular domain protein was per-
formed on 14% Tris-glycine polyacrylamide gels (Invitrogen)
2348 Human Vaccines & Immunotherapeutics
using an X-cell mini cell apparatus (Invitrogen) with Mark 12
unstained standards (Invitrogen) under denaturing and both
non-reducing and reducing conditions. The gels were stained
with Coomassie brilliant blue R-250 (GE Healthcare) or Silver
stained. For the Coomassie Stain gels were stained for 30 min
(following an initial boiling) with a filtered solution of 40%
methanol, 10% glacial acetic acid and two Phastgel Blue R tab-
lets (GE Healthcare) per liter. Destaining occurred with two
changes (after boiling) of 5% methanol, 10% glacial acetic acid
for 15 min each. For the silver staining gels were fixed in a 40%
methanol, 10% acetic acid solution for at least 30 min, and then
washed 3 times in 30% ethanol for 15 min each. Gels were then
incubated in a 1X Dithionite solution for 5 min, washed with
ultra-pure water and placed in a silver nitrate solution for 30 min.
Following two washes with ultra-pure water the gel was developed
in a sodium thiosulfate/carbonate solution containing formalde-
hyde for 10 min or until desired bands appeared. The reaction
was stopped with multiple changes of 3.5% acetic acid solution
and ultra-pure water. Gels were scanned using a calibrated den-
sitometer (Bio-Rad) and the gel images acquired. The acquired
images were analyzed using the Quantity One Software (Bio-
Rad) according to manufacturer instructions. Briefly, for each
gel the Quantity One Software was used to create lanes on the
images corresponding to each of the loaded wells. The back-
ground was subtracted using the rolling disk method, with a
rolling disk size of 100 for each lane. Individual lane plots were
created and a band detected using a sensitivity value of 10, a
width value of 2.5, and a size value of 5. The density of each
band was measured using the Quantity One Software. To quan-
tify the protein concentration in each of the samples loaded a
standard curve was generated by loading Sm-TSP-2 standards at
different concentrations ranging from 0.1 to 1.0 mg/mL on the
same gel. The concentrations of the Sm-TSP-2 samples used as
standards were independently measured using A280. The mea-
sured densities of each of the Sm-TSP-2 standards were plotted
against the concentration of protein loaded and the data set fit
to a linear regression (data not shown). The linear correlation
was used to extrapolate the concentration of the protein from
the densities.
Immunoblot
Proteins resolved by SDS-PAGE were transferred to PVDF
membranes using the iBlot Gel Transfer Stacks Nitrocellulose,
Regular (Invitrogen) and the iBlot transfer unit (Invitrogen).
After transfer, the membranes were blocked with 3% dry milk in
TBS-T20 for 1 h at room temperature. The membranes were then
incubated with 1:1000 rabbit or mouse anti-TSP-2 antibodies
for 1 h at room temperature followed by three washes of 5–10
min each in TBST, and by incubation with an AP-conjugated
anti-rabbit or anti-mouse secondary antibody (KPL Inc.) for one
hour. After additional three washes the blots were developed
with BCIP-NBT developing kit (KPL Inc.). Mouse anti-TSP-2
antibodies were generated by immunization of female BALB/C
mice with an independent preparation of Sm-TSP-2. BALB/C
mice were immunized with a dose of 50 μg of Sm-TSP-2/400 μg
Alhydrogel followed by a boost after 28 days. Rabbit anti-TSP-2
antibodies were provided by Alex Loukas. The Sm-TSP-2 used
Volume 9 Issue 11
for antibodies preparation was purified at a laboratory scale
following the same chromatographic steps as described here.
Reverse phase high performance liquid chromatography
A Waters Alliance® 2695 HPLC system coupled with an
in-line photo-diode array detector (Waters) was used with
a Reverse Phase column (YMC-Pack C8, 150 × 4.6 mm I.D.,
S-5 µm, 20 nm) fitted with a Guard (Column YMC Octyl
[C8] S-5 200A). A mobile phase composed of a water-ethanol
gradient, modified by heptafluorobutyric acid as an ion-pairing
agent in a concentration of 0.1% was used at a flow rate of 1
mL/min. Volumes of Buffer A (5% ethanol, 0.1% HFBA in
water) and B (95% ethanol, 0.1% HFBA in water) were mixed in
function of time in order to achieve the specific gradient. Because
the secondary structure of Sm-TSP-2 extracellular domain
recombinant protein carries a positive net charge, anionic ion-pair
reverse phase chromatography is a relevant technique to improve
chromatographic resolution,31 thus facilitating the determination
of protein identity, purity and integrity. The ion-pair reagent who
provided the optimal peak resolution was the Heptafluorobutyric
Acid (HFBA) at a concentration of 0.1%. The spectrum from
210–400 nm was recorded and UV absorbance of the resultant
peaks was monitored at 215 and 280 nm. To determine system
suitability, 20 μg of bovine serum albumin standard (2.0 mg/mL)
(Thermo Scientific) was injected before each sample set. To
measure the amount of protein, a standard curve was generated by
loading Sm-TSP-2 standards from 0.1 to 1.0 mg/mL in 30 μL of
injected volume. The Sm-TSP-2 peak from the extracted 280 nm
channel was integrated using Empower 2 (Waters) software and
a linear regression peak area vs. Sm-TSP-2 concentration was
generated.
Mass spectrometry
MicroMass Q-ToF spectrometer (Waters) was used to deter-
mine the molecular mass of the recombinant Sm-TSP-2 molecule.
The protein was desalted and diluted into formic acid for analy-
sis. Buffer solution was removed by ZipTip® (Millipore) desalting
prior to analysis. The mobile phase for this analysis was 50%
acetonitrile/0.1% formic acid or various ratios of methanol/water
with 0.1% formic acid. For mass spectrometry analysis, 2–3 μL
of the eluted sample was introduced into the mass spectrometer.
The mass spectrometer was controlled and the data processed
using the MassLynx 4.0 operating system. Approximately 100
scans were combined (to average out fluctuations in the electro-
spray), baseline subtracted, smoothed and the centroid of each
observed peak determined. The software was used to calculate
the mass of the protein from two consecutive multiply charged
ions in the spectrum that were manually defined.
N-terminal sequencing
Edman sequencing was used to obtain N-terminal sequencing
data for the recombinant Sm-TSP-2 molecule. This test was per-
formed by the Foundation Biotechnology Resource Laboratory at
Yale University Proteins. The sample was prepared by desalting
www.landesbioscience.com Human Vaccines & Immunotherapeutics
with a ProSorb Sample Preparation Cartridge (Life Technologies),
which binds the protein to a PVDF membrane in the cartridge.
The PVDF in the cartridge was wet with 20 µl of methanol then
200 µl of ultrapure water and 50 µl of sample were added to the
reservoir and pulled through with a wick. An additional 550 µl of
water was added and pulled through to wash the membrane, then
the PVDF was punched out and air-dried. Before the Edman
Sequencing reaction was performed a glass-fiber sample support
(filter) was treated with 20 µl of BioBrene, air-dried, and pre-
cycled in the sequencer. The sequencer was then calibrated with
a 5 µl sample of internal sequence standard. The PVDF from
the proSorb cartridge was feathered with a razor blade for better
reagent flow and loaded in the sample cartridge above the filter.
A blank, calibration standard, and 13 amino acids of sequence
were run. The resulting chromatograms were printed and over-
laid on a light box to assign the amino acid sequence based on
peak increases (and subsequent decreases) from cycle to cycle.
Amino acid analysis
Amino acid analysis (AAA) was completed on the Sm-TSP-2
extracellular domain to correlate the protein content determined
by A280 using the theoretical extinction coefficient with amino
acid sequence. This test was performed by the Foundation
Biotechnology Resource Laboratory at Yale University. For AAA
analysis, the sample was hydrolyzed into free amino acids using
acid hydrolysis. A 16 h hydrolysis at 115 °C in 100 µL of 6 N
HCl, 0.2% phenol and 2 nmol norleucine, used as an internal
standard, was performed. After hydrolysis, the sample was dried
and then dissolved in 100 µL of Beckman sample buffer contain-
ing 2 nmol homoserine (second internal standard that indepen-
dently monitors sample transfer onto the analyzer). The amino
acid analysis was performed on an L-8900 PH amino acid ana-
lyzer (Hitachi). The analyzer uses an ion-exchange column with
a pH and temperature gradient to separate the amino acids, and
then continually react with ninhydrin for detection at 570 nm
and 440 nm. The analyzer was calibrated during each set of sam-
ples with a 400 pmole mixture of amino acids and was operated
via the manufacturer’s programs and with the use of their buffers.
EZChrome Elite (Hitachi) software was used to run the analyzer
and collect and analyze the data. The raw amino acid nmole data
were then tabulated in a spreadsheet to calculate µg protein/pep-
tide, mole percent, and the predicted number of residues of each
amino acid. The protein concentration was calculated from the
quantity of one or more well recovered amino acids present in the
sample or by summation of total amino acids recovered.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Supplemental Materials
Supplemental materials may be found here:
www.landesbioscience.com/journals/vaccines/article/25787
2349

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