Protein Expression and Purification 134 (2017) 72e81

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Protein Expression and Purification

journal homepage: www.elsevier.com/locate/yprep

Process development for production and purification of the

Schistosoma mansoni Sm14 antigen
Leonardo Damasceno a, Gerd Ritter b, Carl A. Batt a, *
a
Cornell University-Ludwig Institute for Cancer Research Partnership Laboratory, Ithaca, NY, United States
b
Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan-Kettering Cancer Center, New York, NY, United States

a r t i c l e i n f o a b s t r a c t

Article history: The trematode Schistosoma mansoni Sm14 antigen was expressed in the yeast Pichia pastoris and secreted
Received 28 December 2016 into the culture medium at yields of approximately 250 mg L
1. Sm14 belongs to a family of fatty-acid
Received in revised form binding proteins and appears to play an important role in uptake, transport, and compartmentaliza-
22 February 2017
tion of lipids in S. mansoni and it is a potential vaccine candidate in both humans and domesticated
Accepted 3 April 2017
Available online 5 April 2017
animals. The Sm14 gene was codon-optimized for expression in P. pastoris, and placed under tran-
scription of the strong methanol inducible AOX1 promoter. Mutþ transformants were selected and used
in fed-batch cultivation using a 2.5L fermenter equipped with an on-line methanol control system in
order to maintain constant methanol levels during induction. Optimal conditions for the expression of
Sm14 by P. pastoris were found to be: dissolved oxygen at 40%, temperature of 25  C, pH 5.0, and a
constant methanol concentration of 1 gL-1. Our results show that a correctly processed Sm14 was
secreted into the culture medium at levels of approximately 250 mg L
1. Sm14 from clarified culture
medium was purified using a two-step procedure: anion-exchange chromatography followed by hy-
drophobic interaction chromatography, resulting in >95% purity with a final yield of 40% from the
starting cell culture medium. This product has been tested in preliminary clinical trials and shown to
elicit an antibody response with no adverse reactions.
© 2017 Elsevier Inc. All rights reserved.

1. Introduction Chemotherapy remains the foundation for treating schistoso-

Schistosomiasis is one of the most prevalent parasitic infections
and, among other parasitic diseases, is second only to malaria in
terms of morbidity [1,2]. The etiological agents of the disease are
trematodes from the genus Schistosoma. Schistosoma mansoni,
which is the most widely studied, causes hepatic and intestinal
schistosomiasis, S. haematobium causes urinary schistosomiasis,
and S. japonicum causes the hepatosplenic and intestinal form of
the disease. A recent report shows that schistosomiasis is endemic
in 74 countries and territories, and estimates suggest that over 200
million people are infected and over 700 million are at risk [3]. In
addition, there is increasing evidence linking schistosomiasis to
urinary bladder cancer, usually high-grade squamous cell carci-
nomas [4]. In countries with a high incidence of bladder cancer
such as Egypt, Iraq, and Sudan, urinary schistosome infections are
also endemic [5].
* Corresponding author.
E-mail address: cab10@cornell.edu (C.A. Batt).
miasis, but rapid reinfection leads to frequent retreatment and
emphasizes the need for a more long-term approach [6]. The ex-
istence of partially protective immunity in exposed humans makes
a vaccine the logical complement to drug therapy [7]. A potential
candidate for such a vaccine is the S. mansoni fatty-acid binding
protein (FABP) Sm14, which has been recognized by the World
Heath Organization (WHO) as one of two potential anti-
schistosome vaccine candidates for human phase I/II clinical trials
[8,9]. Recombinant Sm14 (rSm14), produced in E. coli, induced
protective immunity in mice by up to 67% against challenge with
S. mansoni cercariae in the absence of adjuvant [10]. In addition, the
protective immunogenicity of Sm14 has been confirmed when
administered via live-attenuated recombinant Mycobacterium bovis
Bacille Calmette-Gue
rin (BCG) and Salmonella enterica var. Typhi-
murium [11,12].
Previous attempts at expressing Sm14 for vaccine purposes have
been restricted to small-scale cultures of E. coli, with the majority of
the recombinant protein found as inclusion bodies [10]. Tendler
et al. [10] have shown that the antigenicity of Sm14 appears to be

http://dx.doi.org/10.1016/j.pep.2017.04.002
1046-5928/© 2017 Elsevier Inc. All rights reserved.
 L. Damasceno et al. / Protein Expression and Purification 134 (2017) 72e81 73

Fig. 1. Sm14 codon optimization for P. pastoris expression. (A) P. pastoris codon usage from original Sm14 gene. Blue bars represent a 50% threshold for codon usage. (B) Optimized
codon usage for P. pastoris expression. Graphs are representative of the first 50 amino acid residues. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)

dependent on conformational epitopes and refolding of rSm14
produced in E. coli is necessary. This refolding process is very
laborious and usually leads to low yields of correctly folded purified
protein. We therefore chose to evaluate the methylotrophic yeast,
Pichia pastoris as the host for Sm14 expression. This organism has
been widely used for the high-yield expression of various proteins
by secretion into the culture supernatant [13e15]. The most
commonly used approach for heterologous protein expression has
been to express the gene of interest under control of the alcohol
oxidase 1 (AOX1) promoter. The expression cassette is then inte-
grated into the P. pastoris genome via homologous recombination
resulting in clones that either retain their methanol-utilization
ability (Mutþ) or exhibit slow growth on methanol (MutS). A
characteristic of the AOX1 promoter is that it is strongly repressed
in cells grown on glycerol, but is induced over 1000-fold when cells
are shifted to a medium containing methanol as a sole carbon
source [15]. Recombinant proteins produced by P. pastoris are
usually properly folded and this organism has no toxic cell wall
pyrogens as found in E. coli, nor contains potentially oncogenic or
viral nucleic acids, as sometimes found in mammalian cells [16,17].
One of the most important factors in protein expression is
adaptation of the codon usage of the transcript gene to the typical
codon usage of the host. Genes that are highly expressed in an
organism under translational selection have marked codon usage
bias due to the use of a small subset of codons that are recognized
by the most abundant tRNA species [18]. If a gene contains codons
that are rarely used by the host, expression levels might not be
optimal, leading to limited recombinant protein expression [19]. A
number of studies have shown that codon optimization can greatly
improve recombinant protein expression levels in P. pastoris and
other organisms [19e24]. Therefore, in this study the Sm14 gene
was codon optimized for expression in P. pastoris.
Here we report the successful expression of Sm14 in a bench-top
fermenter using P. pastoris with a Mutþ phenotype. Sm14 was
secreted in a correctly processed soluble form, at levels of
approximately 250 mg L
1. Although a fraction of the Sm14
74 L. Damasceno et al. / Protein Expression and Purification 134 (2017) 72e81
Fig. 2. Sequence comparison between the native and codon optimized (synthetic) Sm14 genes.
produced by P. pastoris has some form of glycosylation, we describe
a two-step purification protocol where a homogeneous preparation
of non-glycosylated Sm14 is obtained with a recovery of 40% of the
protein secreted into the culture medium.
2. Materials and methods
2.1. Strain, plasmids and culture conditions
P. pastoris strain X33 and plasmid pPICZaA were purchased from
Invitrogen Corp. (Carlsbad, CA, USA), and used in this study.
For general growth, cells were grown in either YPD [1% (w/v)
yeast extract, 2% (w/v) peptone, and 2% (w/v) glucose] or BMG
[100 mM potassium phosphate, pH 6.0; 1.34% (w/v) yeast nitrogen
base without amino acids, 4  10
5% (w/v) biotin; and 1% (v/v)
glycerol].
2.2. Construction of expression plasmid and strain
Codon optimization of the Sm14 gene for P. pastoris expression
was done using Graphical Codon Usage Analyzer software ver. 2.0
(http://gcua.schoedl.de/) [25]. Codons were changed based on
codon usage frequency with a threshold of at least 50% usage. The
codon optimized Sm14 gene was then synthesized de novo by In-
tegrated DNA Technologies (Coralville, IA). This gene was cloned in-
frame with the S. cerevisiae a-mating factor secretion signal under
the control of the AOX1 promoter in pPICZaA, resulting in pPICZaA-
Sm14. P. pastoris X33 was transformed by electroporation, accord-
ing to Invitrogen Corp. (Pichia expression Kit, Version M), with
PmeI-linearized pPICZaA-Sm14. Transformants were selected on
YPD plates containing 18.2% (w/v) sorbitol and 100 mg L
1 Zeocin
(Life Technologies).
2.3. Fermentation conditions
Fermentation experiments were carried out as previously
described, with minor modifications [13]. Briefly, a single
P. pastoris-Sm14 colony was used to inoculate 100 mL of BMG in a
1 L baffled flask, and incubated at 30  C until the culture reached an
OD600 of 10.0. This culture was then used to inoculate the fermenter
medium.
Fermentations were carried out in a 2.5L working volume
bench-top fermenter using a Bioflo 3000 (New Brunswick Scientific
Co., Inc.) interfaced with AFS-Biocommand Bioprocessing software
version 2.6 (New Brunswick Scientific Co., Inc.), for data acquisition
and supervisory control. A starting volume of 1.1L of modified basal
salts medium [0.23 gL
1 CaSO4-2H2O, 4.55 g L
1 K2SO4, 3.73 g L
1
MgSO4-7H2O, 1.03 g L
1 KOH, 6.68 mL L
1 H3PO4, 5% (v/v) glycerol]
and 0.5 mL Antifoam 204 (Sigma, St. Louis, MO) were sterilized
inside the reactor. Ammonium hydroxide [15% (v/v)] was used as a
pH control agent and nitrogen source. The pH was measured with
an Accumet pH electrode (Fisher Scientific). PTM1 trace salts
(24 mM CuSO4, 0.53 mM NaI, 19.87 mM MnSO4, 0.83 mM Na2MoO4,
0.32 mM boric acid, 2.1 mM CoCl2, 0.15 mM ZnCl2, 0.23 M FeSO4,
 L. Damasceno et al. / Protein Expression and Purification 134 (2017) 72e81 75

Fig. 3. Sm14 secretion from P. pastoris grown at different pH values. (A) SDS-PAGE analysis of fermentation samples from induction phase at different pH values. For each pH value,
induction samples at 24, 36, and 48 h were loaded. Lane C ¼ 200 ng of purified Sm14. Position of Sm14 is indicated by arrow. (B) Sm14 concentration at different pH values and
induction time points. Quantification of protein concentrations was done using image densitometry. All samples were normalized for cell mass prior to loading on SDS-PAGE.

0.82 mM biotin) were aseptically added at 4.35 mL L
1 after ster-
ilization and prior to inoculation.
A fermentation temperature of 25  C and the pH value (pH
values from 3 to 6 were examined; see further) were kept constant
throughout batch and induction phases. Dissolved oxygen (D.O)
was maintained at 40% of saturation and was controlled by a D.O
cascade of agitation (maximum of 1000 rpm) followed by supple-
menting with pure oxygen as needed. Oxygen concentration was
measured with an InPro6110/220 electrode (Mettler-Toledo Gmbh,
Germany).
At the end of the batch phase, indicated by a sharp rise in D.O,
the culture was then induced with a methanol feed (100% methanol
with 12 mL PTM1$L
1 methanol) and methanol concentration was
kept constant at 1 g$L
1 with an automated on-line control [13,26].
Samples were taken throughout the induction phase and wet cell
weight (WCW) was used as a measure of cell density. For each time
point, a 1.0 mL sample was centrifuged at 13,000  g for 5 min. The
cell pellet was weighed and the supernatant was stored at
20  C
76 L. Damasceno et al. / Protein Expression and Purification 134 (2017) 72e81
600
500
400
WCW (g/L)
300
200
100
0
0 5 10 15
Fig. 4. P. pastoris biomass accumulation during Sm14 expression at different pH values. Biomass was measured as wet cell weight (WCW).
for further analysis.
2.4. Protein detection and quantification
Culture supernatant samples and chromatography fractions
were analyzed by electrophoresis on 12% SDS-polyacrylamide gels
under denaturing conditions according to standard protocols. For
culture supernatants, 20 mL of cleared supernatant were used for all
SDS-PAGE analyses, unless noted otherwise. The gels were either
stained with a Coomassie-based stain (SimplyBlue SafeStain, Life
Technologies) or silver stained using a SilverQuest kit (Life Tech-
nologies) for total protein visualization. Glycoprotein staining was
carried out in gels using a Pro-Q Emerald 300 Glycoprotein Gel
Stain kit following manufacturer's instructions (Molecular Probes).
Concentrations of Sm14 separated on SDS-PAGE and Coomassie-
stained were estimated using image densitometry software, ImageJ
(http://rsb.info.nih.gov/ij/) as previously described [13]. Sm14
standards were quantified by amino acid composition analysis at
Alphalyse (Palo Alto, CA). Protein identification was done using
mass spectrometry on a MALDI-TOF/TOF 4700 Analyzer (Applied
Biosystems) at the BioResource Center Proteomics and Mass
Spectrometry Facility (Cornell University, Ithaca, NY).
2.5. Purification of Sm14
The fermentation supernatant after induction was clarified
through a 0.22 mm membrane filter and diafiltered against 20 mM
Tris-Cl pH 8.0 for anion-exchange chromatography. All chroma-
€
tography steps were done on an AKTA Explorer (GE Healthcare).
Anion-exchange chromatography (AEX). Anion-exchange
chromatography was performed using a HiTrap Q Sepharose XL
5 mL column (GE Healthcare). The column was equilibrated with
25 mL running buffer (20 mM Tris-Cl pH 8.0). A 25 mL volume of
pH 6
pH 5
pH4
pH3
20 25 30 35 40 45
Induction Time (h)
diafiltered clarified fermentation supernatant was loaded at
5 mL min
1. After washing the column with 10 mL running buffer,
the protein was eluted at 5 mL min
1, with a linear gradient of
0e190 mM NaCl in running buffer.
Hydrophobic interaction chromatography (HIC). Fractions
containing Sm14 (analyzed by SDS-PAGE) were pooled and
adjusted to 1.5 M (NH4)2SO4 and 50 mM Tris-Cl pH 8.0. A 20 mL
sample was applied to a HiTrap Phenyl Sepharose FF (high sub)
1 mL column (GE Healthcare), previously equilibrated with 25 mL
of running buffer [50 mM Tris-HCl pH 8.0 containing 1.5 M
(NH4)2SO4], at 1 mL min
1. Proteins were eluted in a linear gradient
of 1.5e0 M (NH4)2SO4 in the same buffer.
3. Results and discussion
3.1. Codon optimization of the Sm14 gene
The Sm14 gene sequence used in this study codes for a 14.8 kDa
protein with a Cys/Val substitution at position 62, and was based
on a previously published sequence [27]. The C62V substitution
eliminates intermolecular disulfide bond formation and can lead to
increased secretion and facilitate downstream purification.
For codon optimization of the Sm14 gene, P. pastoris codon us-
age frequency with a threshold of at least 50% usage was considered
(Fig. 1). P. pastoris codons for optimal usage were chosen based on
work published by Nakamura et al. [28] and the Codon Usage
Database (http://www.kazusa.or.jp/codon/). Fig. 2 shows sequence
comparison between the native and codon optimized (synthetic)
Sm14 genes. With codon optimization, a reduction of 3.9% in the
G þ C content of the synthetic gene was achieved. Although we did
not compare expression between native and synthetic genes, Sin-
clair and Choy [29] have shown that reduction in the %G þ C con-
tent of the human glucocerebrosidase gene increased expression
 L. Damasceno et al. / Protein Expression and Purification 134 (2017) 72e81 77

Fig. 5. (A) Anion exchange chromatography profile of induced culture supernatant on a HiTrap Q Sepharose XL column. Elution was done in a linear gradient from 0 to 190 mM NaCl
using 20 mM Tris-Cl/1 M NaCl pH 8. (B) Silver-stained SDS-PAGE shows fractions 1e12 (numbered lanes). These fractions are also highlighted in the chromatogram (shaded area).
M ¼ molecular weight marker; I ¼ starting sample. Sm14 bands are indicated by arrow.

7.5-fold in P. pastoris. In addition, a codon optimized Sm14 gene for
expression in M. bovis BCG had a four-fold increase in expression
compared to the native gene [11].
3.2. Effect of pH on Sm14 production
P. pastoris can tolerate a pH range of 3e7, with minimal effect on
growth rate [16]. However, pH variations have been shown to in-
fluence expression of recombinant proteins during the induction
phase [13,16,30,31]. To find the pH at which Sm14 production was
optimal, we conducted experiments with pH values ranging from 3
to 6 during both batch and induction phases. Before inoculation of
the fermentation medium, the tested pH was set and maintained
for the remainder of the experiment. Temperature also remained
constant throughout the experiments at 25  C. This reduction of
temperature from the conventional growth of P. pastoris at 30  C
has been reported in reducing proteolytic degradation of recom-
binant proteins and increased cell viability [13,32].
As shown in Fig. 3, pH 5 resulted in the highest production of
Sm14, reaching 250 mg L
1 after 48 h of induction (Fig. 3-B). In
addition, there was a reduction in degradation of Sm14 at pH 5
when compared to the other pH values tested (Fig. 3-A). These
findings might be due to increased stability of Sm14 at pH 5, which
would lead to lower protease degradation. At pH 6, Sm14 could not
be detected after 36 h induction, mainly due to degradation
resulting from potentially high protease activity at this pH [31].
Since these results show a correlation between pH and Sm14 pro-
duction, all subsequent fermentation experiments were performed
at pH 5.
We also measured biomass accumulation during the induction
phase to determine if the difference in Sm14 production at different
pHs values was related to a difference in biomass accumulation. At
pHs 4, 5, and 6, there was little difference in biomass accumulation
and growth appeared to plateau after ~30 h around 400 g,L
1
WCW (Fig. 4). Conversely, biomass accumulation was higher at pH 3
and after 48 h induction, reached approximately 530 g,L
1 WCW.
Even though biomass at pH 3 is higher than at pH > 3, protein levels
(mg,L
1) were higher at pHs 4 and 5 (Fig. 3). This shows that
78 L. Damasceno et al. / Protein Expression and Purification 134 (2017) 72e81

Fig. 6. (A) Hydrophobic interaction chromatography profile of pooled AEX Sm14 fractions. Elution was done in a linear gradient from 100 to 0% using 50 mM Tris-Cl pH 8.0. (B)
Silver-stained SDS-PAGE showing fractions 8e13 (numbered lanes). These fractions are also highlighted in the chromatogram (shaded area). M ¼ molecular weight marker. Sm14
bands are indicated by arrow.

P. pastoris cells were more efficient at producing Sm14 at pH 5 and Table 1

Steps and yields in the purification of Sm14.
there was no direct correlation between biomass and protein pro-
duction in this study. Purification step Sm14 (mg)a Sm14 Yield (%)b

1. Cell-free culture medium 2.5 100

2. Buffer Exchange 2.5 100
3.3. Purification of Sm14
3. Q Sepharose XL (AEX) 1.6 64
4. Phenyl Sepharose FF (HIC) 0.94 37.6
A two-step chromatographic procedure was established for the a
Sm14 concentrations were estimated using image densitomery from
purification of Sm14 from culture medium. After cultivation, the Coomassie-stained SDS-PAGE.
supernatant was cleared and dialyzed against 20 mM Tris-Cl pH 8. A b
The percentage yield was calculated on the amount of Sm14 recovered at each
25 mL volume of the dialyzed sample was loaded on a HiTrap™ Q step.
XL 5 mL column (see Materials and Methods for details). The eluted
fractions were analyzed by SDS-PAGE (Fig. 5-B) and Sm14 was
confirmed by N-terminal sequencing (data not shown). The ma-
jority of the partially purified Sm14 was eluted at 40 mM NaCl. As revealed this to be a glycosylated form of Sm14.
shown in Fig. 5, this first purification step removed the majority of For the second purification step, a HiTrap Phenyl Sepharose FF
impurities and enriched for Sm14. In addition to the target protein, (high sub) 1 mL column equilibrated with 50 mM Tris-Cl/1.5 M
a higher molecular weight band of approximately 20 KDa was co- ammonium sulfate at pH 8.0 was used to remove remaining im-
purified. N-terminal sequencing and glycoprotein analysis purities. The anion-exchange fractions containing Sm14 were
 L. Damasceno et al. / Protein Expression and Purification 134 (2017) 72e81 79

Fig. 7. Amino acid sequence of Sm14. Potential glycosylation sites are (Asn59 and Asn101) are underlined.

Fig. 8. Glycoprotein profile of Sm14 expression and purification samples. Figure on left is a SDS-PAGE stained with Pro-Q Emerald 300 for glycoprotein detection. Figure on right is
the same SDS-PAGE stained with a Coomassie-based stain for protein detection. M ¼ molecular weight marker. Lane 1 ¼ Cleared culture supernatant at 48 h induction. Lane
2 ¼ Pooled AEX fractions containing Sm14. Lane 3 ¼ Pooled HIC fractions 8e13 (see Fig. 6-A). Lane 4 ¼ Pooled HIC fractions 14e15 (see Fig. 6-A). Lane 5 ¼ Cleared culture su-
pernatant at 24 h induction. Sm14 is indicated by arrow. Glycosylated Sm14 is indicated by dash.

Fig. 9. Partial spectrum of a Sm14 digest analyzed by MALDI-TOF/TOF PSD. This spectrum shows part of the Sm14 sequence FGEEFDEKTSDGR (see Fig. 7 e bold/italicized letters).
80 L. Damasceno et al. / Protein Expression and Purification 134 (2017) 72e81
pooled and adjusted to the same buffer used to equilibrate the HIC
column. A 20 mL sample was loaded on this column and eluted
fractions analyzed by SDS-PAGE (Fig. 6). With this purification step,
Sm14 was eluted at approximately 50% of the ammonium sulfate
gradient, and SDS-PAGE indicated high purity of this protein (Fig. 6-
B), removing the glycosylated form of Sm14 (Fig. 8). A 37.6% of
protein recovery was obtained by this two-step purification scheme
(Table 1).
3.4. Characterization of Sm14 expressed by P. pastoris
Sm14 has two potential glycosylation sites (Asn59 and Asn101)
(Fig. 7), and for its intended use as a vaccine against schistosomi-
asis, and potentially bladder cancer, glycosylation can interfere
with the antigen's immunogenicity. In addition, all of the immu-
nogenicity and toxicity tests done to date with Sm14 were done
using an E. coli-produced antigen, which also lacks glycosylation
[33,34]. Therefore, the first step in characterizing the Sm14 pro-
duced by P. pastoris was to look for potential glycosylation of the
final purified product.
Glycoprotein staining of samples from the different steps in
production and purification revealed that the final purified product
was devoid of glycosylation species of Sm14, as well as any addi-
tional glycosylated proteins naturally expressed by the host (Fig. 8-
Lane 3). This result shows the efficiency of the purification scheme
employed in removing any potentially toxic glycosylated proteins.
Purified Sm14 was also tested for identity using mass spec-
trometry on a MALDI-TOF/TOF (Fig. 9). Results corroborated with
those from N-terminal sequencing of the protein. Together, all of
these results indicate the potential use of Sm14 in vaccine
preparations.
The Sm14 produced in P. pastoris is an excellent candidate for
providing protection against S. mansoni. Clinical trials have been
initiated for the use of this P. pastoris derived Sm14 in both animals
and humans [33]. The Pichia produced SM14 antigen immunized
with GLA adjuvant induced a broad IgG response with no adverse
effects outside of self-limited reactions at the site of injection [34].
The Sm14 antigen in conjunction with the Sm29 antigen has been
reported to provide significant protection against S. mansoni in
mice [35]. In this latter case, the antigen prepared from recombi-
nant E. coli carries an endotoxin burden as well as the complications
from purification of the antigen from the host cells. The P. pastoris
platform has significant advantages of ease-of-use and also the
absence of endotoxin.
Acknowledgments
The authors would like to acknowledge the support of the
Ludwig Institute for Cancer Research. We also acknowledge the
contributions of Andrew Simpson, Miriam Tendler and Celso
Ramos.
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