Egypt. J. Exp. Biol. (Bot.), 10(1): 27 – 35 (2014)
© The Egyptian Society of Experimental Biology
RESEARCH ARTICLE
Sabah Mohamed
Khaled Mahmoud
HIGH
LEVEL
EXPRESSION
HANSENULA POLYMORPHA
OF
ABSTRACT:
The pharmaceutical production of the key
therapeutic protein erythropoietin is largely
dependent on the establishment of a
successful heterologous expression system.
This study reports the successful expression
of the recombinant human erythropoietin in
the
methylotrophic
yeast
Hansenula
polymorpha at a significantly high level (265
mg/l). Although erythropoietin was previously
expressed in a wide variety of mammalian,
bacterial and fungal-based systems, this is
the first time this key hormone is expressed in
the industrially important H. polymorpha. The
significantly high expression level and the
convenience of this organism for the industrial
production of recombinant proteins make the
development of a stable H. polymorpha
erythropoietin transformants an essential
improvement, in the exploitation of this key
protein by the pharmaceutical industry. The
expressed protein was successfully purified
and characterized; confirming its structural
and functional properties, while an industrial
scale development of the system is under
development.
KEY WORDS:
EPO,
Erythropoietin,
H.
polymorpha,
Recombinant proteins, Glycosylated
CORRESPONDENCE:
Khaled Mahmoud
Botany Department, Faculty of Sciences, Ain
Shams University, Cairo, Egypt
Current address:
Leeds Institute of Molecular Medicine, St.
James University Hospital, LS9 7TF, UK
E-mail: bmbkhm@leeds.ac.uk
Sabah Mohamed
Botany Department, Faculty of Sciences, Ain
Shams University, Cairo, Egypt
ARTICLE CODE: 03.02.14
ISSN: 1687-7497
RECOMBIN ANT
ERYTHROPOIETIN
IN
INTRODUCTION:
Erythropoietin (EPO) is an essential
human glycoprotein produced in the renal
fibroblasts, the hepatocytes and the neuronal
cells. It acts as the prime haematopoietic
growth factor; as it plays the principle role in
regulating red blood cells production. An
imbalance in EPO levels can lead either to
anaemia or polycythemia. The recombinant
EPO is used widely for treating cancer and
anaemia patients, especially those with
chronic kidney disease and chemotherapyrelated anemia (Henry and Spivak, 1995; Duh
et al., 2008; Testa, 2010; Chateauvieux et al.,
2011; Bunn, 2013).
Endogenous human EPO is a complex of
165 amino acids which forms a mature 30 kDa
protein with four glycan side chains attached
to it; three located at asparagine 24, 38, and
83, and the fourth is located at Ser-126. In
total, the glycosylation elements comprise
about 40% of the total mass of the protein as
the polypeptide accounts for 18 kDa only
(Jelkmann, 2004). Like other glycoprotein
hormones, EPO exists as a mixture of
isoforms differing mainly in their carbohydrate
structures (Storring and Gaines Das, 1992).
This is because the glycosylation of the
polypeptide moiety is of a post-translational
nature which is influenced by the type of the
cell in which the protein is synthesized and
the physiological factors at the time of the
synthesis.
In
addition,
the
isoforms
composition of an EPO preparation is usually
affected by the purification method which can
also affect its folding (Goto et al., 1988).
Homologously expressed EPO is often
purified in minute quantities from very large
volumes of the urine of the anaemic patients
(Espada and Gutnisky, 1970) or of the plasma
of the anaemic sheep (Goldwasser and Kung,
1971). In 1985, Jacobs et al. isolated the
human EPO cDNA and demonstrated its
heterologous expression in a mammalian host
system. Since then, several patents protected
the production of various EPO isoforms with
similar pharmacological properties (Lin, 1990;
W ellesley et al., 1990; Elliott and Byrne,
2007; Carcagno et al., 2009; Pecker, 2011;
Calo-Fernández and Martínez-Hurtado, 2012).
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28
Egypt. J. Exp. Biol. (Bot.), 10(1): 27 – 35 (2014)
Currently, the recombinant EPO biosimilars (genetically-engineered forms of
erythropoietin) represent the largest market of
a class of biopharmaceuticals; with global
sales of over 13 billion USA dollars per
annum (GBI Research, 2012; Kalorama
Information, 2013). The key patents for the
first generation of EPOs have expired in 2009
and 2010 which opened the market for many
similar analogues to be produced. The
principle step towards any commercial
exploitation of EPO is to establish a reliable
expression system to manufacture the protein
at an industrially viable scale.
W hile most of the recombinant EPO is
produced in mammalian cells-based systems
(Lin et al., 1985), non- glycosylated EPO is
produced in bacteria (Lee-Huang, 1984; Bill et
al., 1995; Nagao et al., 1997). On the other
hand, the baker’s yeast, the methylotrophic
yeast and the soil fungi-based expression
systems
enabled
the
expression
of
recombinant EPO with glycosylation profiles
different from the mammalian cell-produced
forms (Elliott et al., 1989; Celik et al., 2007;
Vats
and
Padh,
2007).
In
addition,
erythropoietin cysteine analogs were recently
expressed in Pichia pastoris (Maleki et al.,
2012).
Naturally, eukaryotic expression hosts
attach oligosaccharides to the side chain of
the asparagine residues existing in specific
order within the amino acid chain of the
protein. Humanized mammalian host cells
usually glycosylate heterologously expressed
proteins by adding a tri-mannose linked to a
galactose and a sialic acid, whereas yeasts
usually hyperglycosylate the proteins by
adding 100 or more of mannose sugars.
Despite
this,
the
yeast-based
protein
expression systems carry many advantages
over the mammalian-based systems; including
lower overall costs, easier growth conditions
and much higher expression levels. Because
of this, the yeast-based expression systems
are usually preferred for the industrial
production of EPO biosimilars as well as
many other recombinant human proteins
despite the over-glycosylation issue (Goeddel,
1990; Mattanovich et al., 2012).
In order to overcome the glycosylation
differences
between
the
mammalian
expression systems and the yeast-based
systems, metabolically engineered yeast
strains were used, in which the yeast
glycosylation pathway was altered to mimic
the
human
glycosylation
profile.
Erythropoietin was expressed in engineered
strains of the methylotrophic yeast Pichia
pastoris where its heavy glycosylation genes
were knocked out and the genes of the human
glycosylation were introduced to confer a
humanized glycosylation profile on the
expressed proteins (Hamilton et al., 2006).
Alternatively,
in
vitro
post-purification
ISSN: 1687-7497
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processing methods could be used to tailor
the yeast-expressed recombinant proteins to
the profile of their mammalian counterparts
(Maras et al., 1997).
Hansenula polymorpha is a GRAS
organism (Generally Regarded as Safe) and
belongs to a group of yeasts capable of using
methanol as a sole source of carbon and
energy and so are referred to as the
methylotrophic yeasts (Gellissen, 2000). A
multitude of expression vectors is available
for use in H. polymorpha where the
recombinant protein expression can be
controlled via a carbon source dependent
repression/derepression/induction mechanism
conferred by the inherent properties of the
promoter used (Gellissen et al., 1992; Esser,
2003).
This yeast is relatively simple to
cultivate and scale-up to thousands of litres in
growth tanks. High yields of heterologously
expressed proteins are achieved through high
gene copy integration, fast growth to high cell
density, and an exclusive library of strong
promoters.
Hansenula polymorpha is also able to
secrete the protein products directly in the
culture medium, a fact that renders the whole
process of downstream processing easier and
less costly. The ability of this organism to
survive in a wide pH range, from 2.5 to 6.5,
makes it a versatile protein factory which is
exploited in the production of various
proteins, each of which requires a very
different optimum pH value throughout the
production process (Kunze et al., 2009).
Overall, high yields of recombinant proteins
can be produced in H. polymorpha at a lowcost. In fact, this organism holds the record in
yeast-derived protein production, with a
productivity of more than 13 g/l in phytase
production (Mayer et al., 1999; Sudbery,
2003).
Amongst the methylotrophic yeasts, P.
pastoris and H. polymorpha are particularly
efficient for commercial-scale recombinant
protein production. These two species are
close relatives; in fact the AOX1 promoter of
P. pastoris is the analogue of the H.
polymorpha
MOX
promoter.
However,
although both promoters are responsive to
methanol, in P. pastoris, alcohol oxidase is
rapidly and strongly induced in response to
methanol induction while in H. polymorpha,
methanol oxidase may be induced either by
methanol or by glycerol de-repression. This is
a particularly significant advantage for using
H. polymorpha in applications involving largescale productions, since it circumvents the
potential hazards associated with the use of
methanol (Gellissen et al., 1992; Gellissen,
2000).
P. pastoris expression vectors are
designed for homologous recombination and
integration at the AOX or HIS4 locus, whereas
http://www.egyseb.org
Mohamed & Mohamed, High Level Expression of Recombinant Erythropoietin in Hansenula Polymorpha
those used in H. polymorpha can integrate by
non-homologous recombination at random
loci. Expression vector copies integrated in P.
pastoris are usually limited to <10, whereas
up to 150 copies can integrate in H.
polymorpha.
Since protein expression is
correlated with gene dosage in these
organisms,
it
is
possible
to
achieve
significantly
higher
expression
in
H.
polymorpha despite the fact that the AOX
promoter of P. pastoris is the strongest
promoter. It is also possible to achieve more
control over the level of production by
regulating the copy number.
W hen plasmids are integrated at the
AOX locus, the gene encoding the alcohol
oxidase is disrupted; once the cells are
transferred to methanol their growth is
significantly retarded. The growth rate of H.
polymorpha remains rapid before and after
induction. In addition, H. polymorpha is a
more thermotolerant organism than is P.
pastoris, easily withstanding temperatures up
to 43°C (Sudbery, 2003).
In this study, we have generated stable
H. polymorpha transformants expressing the
recombinant human erythropoietin at levels
significantly higher than those reported from
other expression systems. The protein was
purified and characterized confirming its
biochemical and functional properties. Scaling
up of EPO production in H. polymorpha in a
fermenter-based set up is well underway.
MATERIAL AND METHODS:
Strains:
Hansenula polymorpha strain 201322 was
obtained from the ATCC. Escherichia coli
strain XL10-Gold® was obtained from Agilent.
Constructs:
Erythropoietin open reading frame construct
was obtained in plasmid pReceiver-Y01 from
GeneCopoeia Inc. Vector pGAPZα was
purchased from Invitrogen.
Reagents and equipments:
Advantage® HD Polymerase Mix, used for
polymerase
reaction-based
amplifications,
was obtained from Clonetech. In-Fusion 2.0
Dry-Down PCR Cloning Kit used for cloning
amplified PCR products was purchased from
Clonetech. Veriti® 96-W ell Thermal Cycler,
Applied Biosystems, was used for thermal
cycling
of
PCR
and
other
enzymatic
incubations. ReadyAgarose Gels and the
ReadySub-Cell GT cell system and buffer
reagents from Bio-Rad were used for nucleic
acids
electrophoresis.
QIAquick
PCR
Purification Kit, QIAquick Gel Extraction Kit
and Plasmid preparation kits from QIAGEN
were used for PCR reaction purification, DNA
gel extraction and plasmid preparations
respectively. All restriction and modification
enzymes were purchased from New England
Bio-labs. Gene Pulser MXcell Electroporation
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29
System,
Bio-Rad,
was
used
for
electroporations.
Innova
4900
incubator
shaker, New Brunswick, and SANYO MIR
Standard Incubator were used for cultures
incubations. Eppendorf centrifuges 5702 R
and 5810R and Avanti® J-26 XP Centrifuge
were used for routine centrifugation and
culture harvest. Corning® 500 ml Erlenmeyer
flask polycarbonate and, PYREX® FernbachStyle Culture Flask with Baffles and Phenolic
Screw Cap, from CORNING, were used for
liquid cultures. BCA Protein Assay Reagent
purchased from Thermo Scientific, was used
for determination of protein concentration.
Cogent® µScale TFF System, from Millipore,
was
used
to
concentrate
EPO
from
supernatants. Criterion Precast Gel System
and buffer reagents from Bio-Rad were used
for
protein
electrophoresis.
Protein
Deglycosylation Mix, New England Biolabs,
was used for protein deglycosylation. Factor
Xa Cleavage Capture Kit, EMD4Biosciencs,
was used for removing the signal sequence
from the expressed protein. Mouse Anti-Myc
tag Monoclonal Antibody, HRP Conjugated,
from abCam, was used in W estern blot
analysis.
iBlot®
W estern
Detection
Chemiluminescent
kit
(anti-Mouse),
Invitrogen, was used for W estern blot
analysis. Cell culture reagents were all
purchased from Becton, Dickinson and
Company (BD). All other laboratory chemicals
were purchased as molecular biology grade
from Sigma. W ater used in reagents
preparations, as well as cell culture was
ultrapure
grade
obtained
from
Milli-Q
Advantage A10 Ultrapure W ater Purification
System, Millipore.
EPO expression in H. polymorpha:
Competent H. polymorpha cells were
prepared and transformed according to Faber
et al. (1994) using the Gene Pulser MXcell
Electroporation System from Bio-Rad. In brief,
a single cell grown on the non-selective YPD
medium (1% Yeast extract, 1% Bactopeptone, 1% glucose, 2% agar) at 37 o C, was
used to grow a fresh overnight culture in
liquid YPD at 37 o C with 250 rpm orbital
shaking. A 100-fold dilution of the growing
cells was made in 200 ml fresh, pre-warmed
(37 o C) YPD medium. Cells were grown at 37 o
C with 250 rpm orbital shaking until the
optical density of the culture reached 1.3 at
663 nm. Cells were then harvested by
centrifugation at 3000 g for 10 minutes and
cell pellet was resuspended in 50 ml of 50mM
potassium phosphate buffer pH 7.5 containing
25 mM dithiotreitol (DTT). Re-suspended cells
were incubated at 37 o C for 10 minutes before
washing twice in 200 ml of the electroporation
buffer STM (270 mM sucrose, 10 mM TrisHC1 pH 7.5 and 1 mM MgC1 2 ).
Cells
were
harvested
again
by
centrifugation at 3000 g and finally resuspended in 1 ml of the STM buffer. 100 µ l
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Egypt. J. Exp. Biol. (Bot.), 10(1): 27 – 35 (2014)
fresh aliquots were used for electroporation
with 5 µ g of the gel purified KpnI-linearized
pGAPURAEPO1 in a pre-chilled electroporation
cuvette. The Gene Pulser MXcell was used to
deliver a 5 mille second pulse using 7.5
kV/cm; 50 µ F and 120 Ohm electric field to
the cells/DNA mixture. Immediately following
the pulse, 1 ml of room temperature YPD
medium was added and the cells suspension
was then incubated for 1 h at 37 o C without
shaking. Cells were harvested (5 min, 3000
g), washed once with 1 ml YND selective
medium (0.67% Bacto yeast nitrogen base
without amino acids, 1% glucose) then spread
on selective YND plates (0.67% Bacto yeast
nitrogen base without amino acids, 1%
glucose+ 100 µ g/ml Zeocin, 2% agar) and
incubated at 37 o C. Surviving transformants
started to grow after three days and were left
to grow further for another 2 days. The
transformation
frequency
was
223
transformants/µ g of DNA and 20 fast growing
colonies were selected and re-streaked three
times (each time 4 days at 37 o C) on selective
YND
plates
until
5
fastest
growing
transformants were finally selected for liquid
culture expression.
A single cell colony from each of the
selected fast growing transformants was used
to inoculate a 10 ml YPD medium in a 500 ml
Erlenmeyer flask for 18 hours at 37 o C with
250 rpm orbital shaking. The 10 ml culture
was then used to inoculate a 300 ml YPD
medium in 2.8 L PYREX® Fernbach-Style
Culture Flask with Baffles and Phenolic Screw
Cap. Cultures were incubated at 37 o C with
300 rpm orbital shaking for 5 days. The EPO
construct was designed so that the α
secretion factor is in frame with the EPO
protein which enables the secretion of the
protein directly in the expression medium.
This
will
facilitate
the
post-expression
concentration and purification of the protein.
Cells were then harvested, (10000 g for 10
minutes) and the supernatant was collected
and kept on ice for processing.
RESULTS AND DISCUSSION:
Design
and
manufacture
of
the
recombinant EPO expression construct:
A range of auxotrophic H. polymorpha host
strains is available which can be transformed by
complementing
their
deficiencies
(positive
control). The ATCC H. polymorpha strain 201322
is a uracil deficient as it lacks the URA3 gene
which
encodes
orotidine-5’phosphate
decarboxylase (ODCase), an enzyme required
for the biosynthesis of uracil. Loss of ODCase
activity leads to a lack of cell growth unless
uracil or uridine is added to the media.
Transforming a URA3 deficient H. polymorpha
with plasmids containing the URA3 gene enables
maintaining these plasmids in an autonomous
multicopy
state
within
the
organism
(Roggenkamp et al., 1986). In addition, the
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presence of a heterologous URA3 gene in the
transformation
plasmids
favours
multiple
integration within the genome which results in
stable transformants that can carry <100 copies
of
the
transformation
construct
without
maintaining the selection pressure (Gatzke et al.,
1995; Brito et al., 1999; Esser, 2003; Sudbery,
2003).
The pGAPZα was selected (Fig. 1a), as the
backbone for constructing an expression vector
to produce the recombinant EPO in the
auxotroph ATCC H. polymorpha strain 201322.
Although this plasmid was originally designed for
expressing recombinant proteins in P. pastoris, it
carries elements which function equally well in H.
polymorpha. The expression promoter was the
glyceraldehyde-3-phosphate
dehydrogenase
promoter (PGAP). This is a high level
constitutive
promoter
which
was
used
successfully for recombinant protein expression
in H. polymorpha. In fact, the expression levels
obtained from PGAP were comparable to those
obtained from the famous methanol oxidase
promoter which is usually used for the
recombinant protein expression in H. polymorpha
(Heo et al., 2003). The alcohol oxidase element
of the pGAPZα vector (AOX TT) also functions
well in H. polymorpha since its transcriptional
machinery correctly recognizes and utilizes the
elements of the alcohol oxidase promoter
(Raschke et al., 1996). In addition, pGAPZα
uses Zeocin as a selection marker for both
bacterial and yeast transformants. Zeocin
selection has also proved to be successful in H.
polymorpha (Salomons et al., 2000; Qian et al.,
2009).
The OmicsLink pReceiver-Y01 construct
(Fig. 1b) was used as a source for the EPO
cDNA (ORF), where the fragment was released
by an EcoRI/NotI double digest. The released
fragment (522 bp) was gel purified using the
QIAquick Gel Extraction Kit according to the
manufacturer recommendations. The Invitrogen
vector pGAPZαA (Fig. 1a) was also EcoRI/NotI
double digested and gel purified before ligated
with the EPO cDNA using T4 DNA ligase.
Ligated vector was transformed into XL10-Gold®
E coli competent cells by heat shock and the
transformants were selected on 100 µg/ml
Zeocin LB agar plates (1% Bacto Tryptone, 0.5%
Bacto Yeast Extract, 1% NaCl, 2% agar) then 10
stable transformants were minipreped and
checked for the presence of the right size EPO
cDNA fragment using EcoRI/NotI restriction
digestion. The sequence and the in frame
alignment of the EPO construct was confirmed
by DNA sequencing and the resulting expression
construct was named pGAPZEPO1 and was
propagated in E. coli on Zeocin selection to
prepare enough DNA using the QIAGEN Midi
plasmid prep kit for subsequent creation of the
final expression construct.
In order to add an extra selection criterion
and encourage the generation of multiple
genome-integrated
constructs,
in
the
H.
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31
Mohamed & Mohamed, High Level Expression of Recombinant Erythropoietin in Hansenula Polymorpha
polymorpha
expression
transformants,
we
inserted the S. cerevisiae URA3 gene (Bonneaud
et al., 1991) into the pGAPZEPO1 construct at
the BglII restriction site. Gatzke et al. (1995)
showed that the copy number of the integrated
plasmids in the transformed H. polymorpha is
dependent on the plasmid marker used for the
selection of the transformants. Since the URA3
gene of S. cerevisiae is expressed at a low level
in H. polymorpha, the selection of transformants
with the highest growth rate positively
discriminates cells with multiple copies of this
marker. In contrast, plasmids bearing the
homologous H. polymorpha URA3 gene usually
integrate into the genome in a single copy. In
order to do this, we used the OmicsLink
pReceiver-Y01x vector (Fig. 1B) as a template to
amplify the S. cerevisiae URA3 segment using
PCR. Forward and reverse oligonucleotide
primers flanking the URA3 segment were
designed carrying the BglII recognition site. The
forward primer was designed so that an extra
restriction site (KpnI) is located 3’ to the BglII
site with the intention that it can be used for
linearization of the final construct (Fig. 1C).
Primers were synthesized and purified before
used in the PCR amplifications. Using the
Advantage® HD Polymerase Mix and the purified
DNA of the vector pReceiver-Y01x, as a
template, a 1.2 kbp URA3 fragment was
amplified and gel purified using the QIAquick Gel
Extraction Kit.
The In-Fusion 2.0 Dry-Down PCR Cloning
Kit was used to clone this 1.2 kbp URA3
fragment into the pGAPZEPO1 construct at the
BglII
site
according
to
manufacturer
recommendations. The generated plasmid was
called pGAPZURAEPO1 and was transformed
into XL10-Gold® E. coli competent cells by heat
shock
according
to
the
manufacturer
recommended procedure.
The bacterial
transformants were selected on 100 µg/ml
Zeocin LB agar plates where 5 stable
transformants were minipreped using the
QIAGEN plasmid miniprep kit, according to the
manufacturer recommendations, and checked for
the presence of the right size URA3 segment by
PCR using the same amplification primers. The
resulting construct (Fig. 1C) was used in
transforming H. polymorpha strain 201322 to
express EPO as described in the following
sections.
Processing and characterization of the
expressed EPO:
The Cogent® µScale TFF system was used
for protein collection and concentration from the
ice-cooled clear supernatant according to
manufacturer
recommendations.
This
concentration process is dependent upon
membrane filtration technology called tangential
flow filtration (TFF). The process uses filtration
membranes with specific cut off threshold to
separate components in a liquid solution based
on their molecular weight and size. A 10 kDa cut
off was used to make sure that the concentration
I ISSN: 1687-7497
process collects all of the recombinant protein
that was expressed in the culture medium. Figure
2 lane C, shows a silver stain of 20 µl of the
concentrated supernatant run on 4-20% precast
gradient Tris-HCl SDS-PAGE. Despite the
presence of some background proteins, the
predominant species is about 36 kDa in size
which is likely to be the glycosylated EPO. The
recombinant EPO construct (Fig. 1C) was
designed to express in frame, at the N-terminus,
with the myc epitobe, as well as a 6 poly-histidin,
tag which will facilitate purification from the
culture supernatant.
Fig.
1.
Constructions
of
the
erythropoietin expression construct.
recombinant
a: Invitrogen pGAPZα vector.
b:
GeneCopoeia
erythropoietin
clone
in
pReceiverYO1x vector.
c:
Recombinant
erythropoietin
expression
construct used to express the protein in H.
polymorpha.
The QIAGEN Ni-NTA Superflow Columns
were then used, according to manufacturer
recommendations, to purify the recombinant
EPO from the concentrated total protein
supernatant, collected from the expression
culture.
The
clear
and
concentrated
supernatant was passed through the buffer
equilibrated columns, under native conditions,
washed twice with the washing buffer before
the protein was eluted with the elution buffer.
The purified EPO was then treated with the
factor Xa protease to remove the N-terminus
α-signal sequence from the native EPO. The
Factor Xa Cleavage Capture Kit was used for
this purpose according to manufacturer
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32
Egypt. J. Exp. Biol. (Bot.), 10(1): 27 – 35 (2014)
recommendations. Purified EPO was mixed
with the Cleavage/Capture Buffer and diluted
factor Xa, incubated at room temperature for
6 hours before factor Xa was removed by
affinity purification of the kit Xarrest Agarose.
The equilibrated agarose was mixed with the
protease reaction with gentle agitation for 15
minutes before the agarose was separated
from
the
clear
buffered
protein
by
centrifugation. Figure 2 lane B is showing the
Ni-column purified EPO run on Coomassiestained 4-20% precast gradiant Tris-HCl SDSPAGE. Despite showing a molecular weight
slightly smaller than the predominant species
in the un-purified culture supernatant (lane
C), the purified EPO is showing a size of 34
kDa. The native mature EPO is known to be
30 kDa in size and the difference is probable
due to over glycosylation, as well as the tags
linked to the protein at it C-terminus (Fig. 1C).
However, the concentration of the purified
protein was estimated by the BCA Protein
Assay Reagent, according to the manufacturer
recommendations.
We
estimated
that
assuming 100% purification efficiency via the
concentration and the Ni-purification, EPO
was produced in H. polymorpha at 265 mg/l of
culture medium. This is about 50 fold the yield
reported before in P pastoris (Celik et al.,
2007) and more than double EPO cysteine
analogs expression under the same AOX
promoter in shake flasks (Maleki et al., 2012).
This significantly high yield may be attributed
to a construct multi-copy integration event
which is known to be associated with the
heterologous recombination of the URA3
marker into the H. polymorpha genome
(Gatzke et al., 1995).
H. polymorpha is
known to be particularly suitable for large
scale
fermenter
recombinant
protein
production (Mayer et al., 1999; Kunze et al.,
2009; Crommelin et al., 2013). This high EPO
expression level potentiates the likelihood of
a successful industrial fermenter scale
production.
After the factor Xa digestion, the purified
EPO
was
deglycosylated
under
nondenaturing conditions by mixing it with the
deglycosylation mix from NEB and incubated
it for 4 hours at 37 o C. The deglycosylated
EPO was then passed through the Ni-columns
to purify it from the deglycosylation mix.
Figure 2 lane A is showing the deglycosylated
purified EPO after factor Xa treatment. The
SDS PAGE migration is reflecting the removal
of the glycosylation as in lane B the signalless recombinant EPO is migrating at about
35 kDa while after deglycosylation (lane A)
the migration of the protein was shifted to just
above 17 kDa. In order to confirm the identity
of the expressed EPO, anti-myc antibodies
were used to interrogate the protein using
W estern blotting. The recombinant EPO
construct (Fig. 1C) was designed so that a
myc epitobe is fused to the mature protein Cterminus. Figure 3 is showing the X-ray
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On Line ISSN: 2090 - 0503
exposure of a chemiluminescence W estern
blot of the α signal-free EPO after and before
deglycosylation. Despite some degradation,
that is likely to result from post-expression
processing and purification, the recombinant
EPO is reacting positively to the anti-myc
antibody detection which confirms the proper
structure of the expressed protein as
designed (Fig. 1C). The W estern blot has also
showed a predominant uniform band migrating
at 18 kDa (lane A) and two closely located
bands (lane B) migrating at 30 and 35 kDa,
respectively.
Fig. 2. SDS-PAGE of the recombinant erythropoietin
expressed in H. polymorpha.
a: purified-deglycosylated EPO after removal of
the signal sequence.
b: purified EPO before deglycosylation.
c: concentrated expression supernatant before
EPO purification.
In order to confirm the identity of these
bands a copy of the Western blot membrane
was used to perform N-terminus protein
sequencing for the three predominant bands
(Fig. 3). The protein bands were electroblotted to the PVDF membrane which was
then stained with Coomassie brilliant blue.
The bands were carefully excised from the
membrane and were sent for N-terminus
protein sequencing service at the Proteome
Factory AG. It was revealed that the 18 kDa
bands on lane A (Fig. 3), as well as the two
closely migrating bands on lane B are sharing
the same N-terminus amino acid sequence of
Met-Gly-Val-His-Glu-Cys-Pro-Ala-Trp-Leu-Trp.
This result confirmed that the factor Xa
digestion to remove the α-signal sequence
from the mature EPO was efficient and the
protein expressed is actually the recombinant
human EPO. It is likely that the two closely
migrating bands, on lane B, are two differently
glycosylated isoforms of the recombinant
EPO, since after the deglycosylation, the
protein migrated as a predominantly single
band on lane A.
http://www.egyseb.org
Mohamed & Mohamed, High Level Expression of Recombinant Erythropoietin in Hansenula Polymorpha
Fig.
33
3. Western blot of the recombinant
erythropoietin expressed in H. polymorpha using
anti-myc antibodies.a: purified-deglycosylated
EPO after removal of the signal sequence. b:
purified EPO before deglycosylation. The Nterminus amino acid sequence is shown next to
each of the major bands.
CONCLUSION:
Hansenula polymorpha proved to be a
suitable expression host for the production of
the recombinant human erythropoietin under
shaking flask conditions. Despite the overglycosylation problem of the synthesized
EPO, the high yield achieved using the URA3
integration construct with the PGAL promoter
carries a great potential for large scale
production due to the significantly high yield.
The generated transformants were maintained
under non-selective conditions which make
them particularly suitable for producing this
pharmaceutically essential protein under
industrial fermentation conditions.
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‫‪35‬‬
‫‪Mohamed & Mohamed, High Level Expression of Recombinant Erythropoietin in Hansenula Polymorpha‬‬
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