High-level soluble expression of a thermostable xylanase

from thermophilic fungus Thermomyces lanuginosus

in Escherichia coli via fusion with OsmY protein
Yilin Le

, Huilei Wang
School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, Jiangsu 212013, PR China
a r t i c l e i n f o
Article history:
Received 11 February 2014
and in revised form 7 March 2014
Available online 18 March 2014
Keywords:
Xylanase
Fusion protein
Soluble expression
Escherichia coli
Thermomyces lanuginosus
a b s t r a c t
A thermostable xylanase is encoded by xynA from fungus Thermomyces lanuginosus. The problem
emerged from overexpression of xynA in Escherichia coli has been the formation of inclusion bodies. Here
we describe the xynA was fused with the hyperosmotically inducible periplasmic protein of E. coli, OsmY.
The fusion protein OsmY-xynA was expressed as almost all soluble form. The soluble expression level of
fusion protein reached 98 6 U/ml when cells containing pET-OsmY-xynA were expressed without IPTG
induction at 37 C. The induction is probably due to auto-induction due to lactose in the medium (Studier
(2005) [21]). The cells harboring pET-OsmY-xynA expressed an activity level about 24 times higher than
that expressed from pET-20b-xynA. Xylanase activity was observed in the extracellular (36 1.3 U/ml)
and the periplasmic (42 4 U/ml) when cells containing pET-OsmY-xynA were induced without IPTG
addition. After the cold osmotic shock procedure followed by nickel afnity chromatography, the puried
fusion protein showed a single band on SDSPAGE gel with a molecular mass of 44 kDa. The puried
fusion enzyme exhibited the highest activity at 65 C and pH 6.0.
2014 Elsevier Inc. All rights reserved.
Introduction
Thermomyces lanuginosus produces a thermostable GF11 endo-
xylanase encoded by xynA gene. This xylanase is free of cellulase
activity, and hydrolyses xylan to produce xylooligosaccharides
with little xylose [1]. These properties make the enzyme attractive
for the industrial application. Escherichia coli is one of the most
extensively used prokaryotic organisms for the industrial produc-
tion of enzyme because of its well-characterized genetics, and its
ability to grow rapidly and at high density on inexpensive sub-
strates [2].
The xylanase gene xynA has been sequenced and cloned into
E. coli as a LacZ fusion protein, but efcient expression was not
obtained [3]. Recently, the DNA sequence of xynA has been opti-
mized, and the expression of the enzyme in E. coli has reached
a high level by using recombinant plasmid pET-20b-xynA. How-
ever, the recombinant enzyme was mainly found in inclusion
bodies, and only a small proportion was soluble and active [4].
It is a common problem that some recombinant proteins will
aggregate to form inclusion bodies in the cytoplasm and/or
periplasm [5]. Inclusion body formation of eukaryotic proteins
in E. coli with many contributing factors: insolubility of the prod-
uct at the concentrations being produced, inability to fold cor-
rectly in the bacterial environment, or lack of appropriate
bacterial chaperone proteins [6]. Many attempts have been made
to improve the soluble expression of recombinant proteins in
E. coli. The formation of inclusion bodies could be decreased by
changing the promoter to regulate the level of expression, con-
trolling the growth conditions (especially the pH of the culture),
controlling fermentation medium, changing the temperature of
induction and enabling secretion into the periplasm, fusing the
target gene to another gene [7].
OsmY has been used as a fusion partner to excrete target pro-
teins into the medium [810]. When fused to OsmY, E. coli alkaline
phosphatase, Bacillus subtilis a-amylase, and human leptin could be
secreted into the medium at high levels [11].
Here we report the construction of vector pET-OsmY-xynA, and
the overexpression of soluble fusion protein OsmY-xynA in E. coli.
Materials and methods
Bacterial strains, plasmids and growth media
E. coli DH5a (TaKaRa, Dalian, China) was used as hosts for gene
cloning. E. coli BL21(DE3) (TaKaRa, Dalian, China) was used as hosts
http://dx.doi.org/10.1016/j.pep.2014.03.004
1046-5928/ 2014 Elsevier Inc. All rights reserved.

Corresponding author. Tel.: +86 511 88796122.

E-mail address: leyilin@163.com (Y. Le).
Protein Expression and Purication 99 (2014) 15
Contents lists available at ScienceDirect
Protein Expression and Purication
j our nal homepage: www. el sevi er. com/ l ocat e/ yprep
for the expression of fusion protein. The cells harboring expression
plasmids were cultured in LuriaBertani (LB)
1
medium supple-
mented with ampicillin (100 lg/ml).
Construction of fusion expression plasmid pET-OsmY-xynA
Based on the nucleotide sequences coding for the OsmY protein
(GenBank accession No. NC_000913.3), the PCR amplication was
carried out by using E. coli DH5a genome as template, with the fol-
lowing primers: 5
0
-GCCGAATTCATGACTATGACAAGACTGAAG-3
0
and 5
0
-GGCGGATCCCTTAGTTTTCAGATCATTTTTAAC-3
0
. PCR was
carried out using the high-delity Pyrobest DNA polymerase (TaKa-
Ra, Dalian, China). PCR products were puried using the QIAquick
PCR purication kit and followed by digestion with BamHI and EcoR
I restriction enzyme(s). The reverse PCR amplication was carried
out by using plasmid pET-20b-xynA [4] as template, with primers
X1 (5
0
-GCCGAATTCTATATCTCCTTCTTAAAGTTAAAC-3
0
) and X2
(5
0
-GGCGGATCCCAGACTACCCCGAACTCTGAAG-3
0
). PCR products
were puried using the QIAquick PCR purication kit and followed
by digestion with corresponding restriction enzyme(s). The di-
gested PCR products were ligated to OsmY at BamH I/EcoR I sites.
Expression and purication of fusion protein
The plasmid pET-OsmY-xynA was transformed into the E. coli
BL21(DE3) by electroporation. The cells carrying pET-OsmY-xynA
were grown at 37 C, and induced for gene expression by addition
IPTG (isopropyl-b-D-thio galactopyranoside). The recombinant en-
zyme was isolated from the periplasm by cold osmotic shock
according to a published protocol [12]. The cells (wet weight
1.5 g) harvested by centrifugation at 6000g for 5 min were re-
suspended in 12 ml of 100 mM TrisHCl containing 20% sucrose
and 1 mM EDTA (pH 8.0), and then pelleted by centrifugation at
8000g for 5 min followed by re-suspension in 5 ml of ice-cold
water for 10 min. After the addition of MgCl
2
to a nal concentra-
tion of 1 mM, the cell suspension was incubated on ice for a further
10 min before being pelleted by centrifugation at 8000g for
10 min at 4 C. The supernatant (5 ml) was loaded onto a 1 ml Hi-
sTrap HP columns (GE Healthcare), washed with 60 mM imidazole
and 0.5 M NaCl in 20 mM TrisHCl buffer (pH 7.9), and eluted with
1 M imidazole and 0.5 M NaCl in 20 mM TrisHCl buffer (pH 7.9).
The pooled fractions were dialyzed into storage buffer containing
1 mM EDTA, and 20% (v/v) glycerol before the enzyme was stored
at 20 C. The SDSPAGE was performed according to standard
procedures. Protein concentration was determined by the Bradford
method using BSA as a standard [13].
Enzyme assays
Xylanase activity was determined by the 4-hydroxybenzoic acid
hydrazide method [14]. Xylan from birch wood (Sigma Aldrich,
Munich, Germany) was used as the substrate. The reaction mixture
comprised of 100 ll 1% (w/v) birch wood xylan in water, 90 ll
phosphate buffer (50 mM, pH 6.0) and 10 ll properly diluted en-
zyme. The reaction was conducted at 65 C for 10 min, and stopped
when 600 ll of 4-hydroxybenzoic acid hydrazide solution were
added into the reaction mixture. The reducing sugar was deter-
mined by reading the absorbance at 410 nm after the test tubes
were incubated for 10 min in boiling water bath and cooled down
on ice. One unit of xylanase activity was dened as the amount of
enzyme releasing 1 lmol reducing sugar per min.
Results
Construction of expression plasmids
The gene encoding the OsmY (including the signal sequence)
was amplied from the genomic DNA of E. coli DH5a, and inserted
into the plasmid pET-20b-xynA (pelB signal sequence was deleted)
[4] at BamH I/EcoR I sites. Newly generated plasmid is designated
as pET-OsmY-xynA (carried an N-terminal OsmY signal sequence).
Target protein xylanase from fungus T. lanuginosus was linked to
the C-terminus of OsmY by a BamH I site sequence. The fusion pro-
tein OsmY-xynA was expressed with a C-terminal His-tag.
Expression level and solubility of the fusion protein OsmY-xynA
The recombinant plasmid pET-OsmY-xynA was isolated, which
was then transformed into E. coli BL21(DE3) for the production of
fusion protein OsmY-xynA using IPTG induction. The xylanase
activity of fusion protein was obtained after induction at different
IPTG concentrations (Fig. 1). Interestingly, xylanase activity pro-
duced by cells containing pET-OsmY-xynA with 0 mM, 0.1 mM,
0.3 mM, 0.5 mM IPTG, was 98 6 U/ml, 6.4 0.2 U/ml, 6.3 0.15
U/ml, 6 0.1 U/ml, respectively. There was a gradual decrease in
xylanase activity upon increasing the IPTG concentration (Fig. 1).
The recombinant cells induced without IPTG addition produced
an activity level 16 times higher than that expressed with
0.5 mM IPTG induction.
Previously, intracellular expression of the xylanase was im-
proved by sequence optimization by site-directed mutagenesis
without changing the protein sequence [4]. But the recombinant
xylanase mainly appeared as inclusion bodies [4]. In the current
study, the expression levels and solubility of the fusion protein ex-
pressed from pET-OsmY-xynA were shown in Fig. 2. The fusion
protein expression level is very high when the fusion gene expres-
sion was induced by the addition of IPTG at 37 C (Fig. 2, line 35),
and only a small proportion was soluble and active (Fig. 2 line 79).
However, the fusion protein was expressed as almost soluble form
when the fusion gene expression was induced without IPTG addi-
tion (Fig. 2 line 2 and 6).
Secretion of fusion protein in E. coli
When pET-OsmY-xynA vector was used to express fusion pro-
tein, the xylanase activities in the extracellular, periplasmic and
cytoplasmic fractions were monitored (Fig. 3). Protein expression
was initiated by the addition of IPTG to a nal concentration of
0.5 mM when the optical density at 600 nm (OD
600
) reached 0.8.
Fig. 1. Total expresion levels of fusion protein OsmY-xynA in cytoplasm, periplasm
and culture medium were observed after induction at different IPTG concentrations.
1
Abbreviations used: LB, LuriaBertani; IPTG, isopropyl-b-D-thio galactopyranoside.
2 Y. Le, H. Wang / Protein Expression and Purication 99 (2014) 15
When E. coli BL21(DE3) was used as host to express fusion pro-
tein in LB medium, a maximal cell density (OD
600
5.3 0.1) was ob-
tained without IPTG addition (Fig. 3). In comparison, a signicantly
higher level of xylanase activity was observed in the extracellular,
periplasmic and cytoplasmic fractions when cells induced without
IPTG addition (Fig. 3).
As shown in Fig. 3a, when the fusion protein was expressed by
the addition of IPTG to a nal concentration of 0.5 mM, most of the
xylanase activity (5.7 0.2 U/ml) was observed in the extracellular
fraction at 24 h post-induction. However, when the fusion protein
was expressed without IPTG induction, most of the activity was ob-
served in the extracellular (36 1.3 U/ml) and the periplasmic
(42 4 U/ml) (Fig. 3b).
Purication of the fusion protein
The fusion protein was puried in two steps: cold osmotic
shock and nickel afnity chromatography (Table 1). The periplasm
fusion proteins were extracted by the cold osmotic shock proce-
dure and furthermore puried with nickel afnity chromatogra-
phy. An analysis by SDSPAGE showed that the recombinant
fusion OsmY-xynA had a molecular mass of about 44 kDa (Fig. 4).
After the enzymes were puried, the specic activity of fusion pro-
tein OsmY-xynA was 739 45 U/mg. In comparison, the puried
xylanase from Aspergillus niger exhibited a specic activity of
808.5 U/mg towards birch wood xylan [15]. The specic enzyme
activity of xylanase from Thermotoga thermarum was up to
145.8 U/mg [16]. XynA from Clostridium cellulovorans had a high
specic activity with birch wood xylan (825 U/mg) [17].
Effects of pH and temperature on enzyme activity and stability
When birch wood xylan was used as substrate, the optimal
reaction of fusion protein occurred at 65 C, pH 6.0 (Fig. 5a and
b). The puried enzyme retained over 80% of its activity after hold-
ing at a pH ranging from 5.8 to 7.8 for 1 h at 60 C (Fig. 5c). The
thermostability was evaluated by determination of the residual
ligating activity after incubating the mixtures (0.042 mg/ml
OsmY-xynA, 50 mM pH 6.0) for various times at 60 C, 65 C,
70 C. Fig. 5d shows the residual activity assayed under standard
reaction conditions.
Table 1
Purication of fusion protein OsmY-xynA from E. coli harboring pET-OsmY-xynA.
Purication step Total protein (mg) Total activity (U) Specic activity (U/mg) Purication fold
Periplasmic fraction 3.3 442.2 134 1.0
Nickel afnity chromatography 0.27 199.5 739 5.5
Fig. 4. SDSPAGE analysis for the expression of fusion protein. lanes: M, protein
markers; 1, total protein of E. coli containing pET-20b(+); 2, total protein and 3,
soluble protein of E. coli containing pET-OsmY-xynA; 4, periplasmic protein
obtained by cold osmotic shock; 5, fusion protein puried after a His-tagged
afnity chromatography.
Fig. 3. The effect of IPTG induction on the cell density and the production of recombinant protein in the E. coli cells harboring pET-OsmY-xynA. When the OD
600
reached 0.8
(denoted as time zero), IPTG was added to cell cultures. (a) with 0.5 mM IPTG induction, (b) without IPTG addition. Symbols: -j- cell density; -D- xylanase activities in the
extracellular; -h- xylanase activities in the periplasmic; -s- xylanase activities in the cytoplasmic fractions.
Fig. 2. SDSPAGE analysis for the expression of fusion protein in pET-OsmY-xynA
after induction at different IPTG concentrations. Lanes: M, protein markers; 1, total
protein of E. coli containing pET-20b(+); 25, total protein of E. coli containing pET-
OsmY-xynA induction with 0 mM, 0.1 mM, 0.3 mM, 0.5 mM IPTG, respectively; 69,
soluble protein of E. coli containing pET-OsmY-xynA induction with 0 mM, 0.1 mM,
0.3 mM, 0.5 mM IPTG, respectively.
Y. Le, H. Wang / Protein Expression and Purication 99 (2014) 15 3
Discussion
To facilitate enzyme production and the industrial use of the
thermophilic xylanase preparations, several xylanase genes from
thermophilic fungi have been cloned and expressed in different
heterologous systems [18,19]. The mature xylanase from T. lanug-
inosus has 196 amino acids, with 31 amino acids encoded by the
codons rarely used in E. coli [3]. In previous study, the xylanase
gene from T. lanuginosus had been optimized for high level expres-
sion in the cytoplasm of E. coli by using pET-20b-xyA, but the en-
zyme was produced at a largely insoluble state, the soluble
expression level of recombinant enzyme was very poor, with a
highest activity of 4.1 U/ml [4].
OsmY of E. coli is the hyperosmotically inducible periplasmic
protein. Many studies have been focused on the excretion of target
proteins into the medium. When fused to OsmY, many recombi-
nant proteins of different origin could be excreted into the medium
[811].
Here, we constructed a vector pET-OsmY-xynA to export fusion
protein. OsmY-xynA was successfully expressed at a high level in a
soluble form when the fusion gene expression was induced with-
out IPTG addition. Interestingly, the majority of the recombinant
proteins were in the insoluble fraction when cells were induced
by the addition of different IPTG concentrations (Fig. 2). SDSPAGE
analysis revealed that the fusion proteins expressed from pET-
OsmY-xynA after induction at different IPTG concentrations were
separated into two adjacent bands, suggesting that some of the en-
zyme molecules were still carrying signal peptide in the periplasm
(Fig. 2). These observations suggested that recombinant fusion
xylanase was not correctly folded when the heterogeneous pro-
teins were expressed in a very high rate after induction with IPTG.
The pET system is the most powerful system used for the cloning
and expression of recombinant proteins in E. coli [20].
Induction is probably due to auto-induction due to lactose in
the medium [21]. It is known that LB medium may be contami-
nated with lactose, and lactose contamination increases back-
ground level of protein expression. Supplementing culture media
with glucose could maintain very low basal expression levels of
T7 RNA polymerase in the kDE3 lysogenic expression hosts used
in the pET System [22,23]. Much lower expression of the target
protein was observed (4.5 0.5 U/ml) when the strain harboring
pET-OsmY-xynA was grown in LB medium supplemented with
1% glucose by incubation overnight at 37 C without IPTG.
In our previous study, the xylanase gene fromT. lanuginosus was
overexpressed in the cytoplasm, but the enzyme was produced at a
largely insoluble state [4]. The improvement of soluble protein is
primarily attributed to the fact that the periplasmic spac provides
a more oxidative environment than the cytoplasm [24]. The fusion
proteins were translocated into the periplasmic space, and enzyme
activity analysis shown that about 43% of enzyme activity was
localized in the periplasmic space (Fig. 3a). A simple osmotic shock
was used to obtain the products (Fig. 4).
Fig. 5. The effects of temperature and pH on the xylanase activity and enzyme stability of OsmY-xynA. (a) The optimal temperature determined by standard assay at various
temperatures (pH 6.0). (b) The optimal pH determined at 65 C in citrate buffer buffer, or phosphate buffer. (c) The pH stability of OsmY-xynA. The remaining activity was
determined after puried enzyme (0.042 mg/ml) was incubated at 60 C for 1 h in 50 mM in citrate buffer buffer, or phosphate buffer. (d) The thermostability of the OsmY-
xynA. The puried enzyme (0.042 mg/ml) in 50 mM phosphate buffer (pH 6.0) was incubated for various durations at 60 C (-N-), 65 C (-d-), 70 C (-j-). Residual activities
were assayed at 65 C in 50 mM phosphate buffer (pH 6.0). The initial activity (739 45 U/mg) was dened as 100%. Data are means standard deviations from three
replications.
4 Y. Le, H. Wang / Protein Expression and Purication 99 (2014) 15
Previous results have shown that the signal peptide of OsmY is
responsible for the secretion into the periplasm, while OsmY is
responsible for the excretion from the periplasm to the medium
[11]. On the basis of xylanase activity, about 37% of enzyme activ-
ity was in the mediumwhen cells were induced without IPTG addi-
tion (Fig. 3).
In summary, while the mechanism for the pET-OsmY-xynA
mediated high-level soluble expression of an aggregation-prone
xylanase in E. coli is not completely understood, this paper offers
an alternative protocol to prevent the inclusion body formation
of a thermostable xylanase from thermophilic fungus.
Acknowledgments
This work was supported by the National Natural Science Foun-
dation of China (Grant No. 31300088), the Natural Science Founda-
tion of the Jiangsu Higher Education Institutions of China (Grant
No. 12KJB180002) and Doctoral Scientic Research Foundation of
Jiangsu University (Grant No. 10JDG117).
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