Purification and refolding of a novel β-agarase from inclusion body of E. coli

Abstract

β-agarase AgaB appears to represent a new family of glycoside hydrolase; it is structurally and functionally different from
other known agarases. In the present study, AgaB was expressed with a temperature-inducible expression system in E. coli BL21 (DE3) as a fusion protein bearing a C-terminal hexahistidine tag. The protein existed mainly in the form of inclusion
body. After being washed and solubilized, AgaB in inclusion body was denatured and purified to electrophoretic purity by immobilized
metal affinity chromatography. The purified AgaB was then refolded using a simple pulse dilution method, and the refolded
AgaB showed a high specific hydrolysis activity of about 1600 units /mg protein. Forty milligrams of refolded pure protein
were obtained from 1L of culture.

Full text

Page 1

Journal of Ocean University of China (Oceanic and Coastal Sea Research)
ISSN 1672-5182, January 30, 2007, Vol.6, No.1, pp.80-84
http://www.ouc.edu.cn/xbywb/
E-mail:xbywb@ouc.edu.cn
Purification and Refolding of a Novel β - Agarase
from Inclusion Body of E. coli
ZHANG Li, LU Xinzhi, HAN Feng, MA Cuiping, and YU Wengong*
Marine Drug and Food Institute, Ocean University of China, Qingdao 266003, P.R. China
(Received March 6, 2006; accepted November 15, 2006)
Abstract β-agarase AgaB appears to represent a new family of glycoside hydrolase; it is structurally and functionally different
from other known agarases. In the present study, AgaB was expressed with a temperature-inducible expression system in E. coli
BL21 (DE3) as a fusion protein bearing a C-terminal hexahistidine tag. The protein existed mainly in the form of inclusion body.
After being washed and solubilized, AgaB in inclusion body was denatured and purified to electrophoretic purity by immobilized
metal affinity chromatography. The purified AgaB was then refolded using a simple pulse dilution method, and the refolded AgaB
showed a high specific hydrolysis activity of about 1600 units /mg protein. Forty milligrams of refolded pure protein were obtained
from 1L of culture.
Key words β-agarase; inclusion body; refolding; immobilized metal affinity chromatography
DOI 10.1007/s11802-007-0080-z

1 Introduction
Agarases produced by agar-degrading bacteria are
classified into two groups based on their modes of action,
namely, α-agarase and β-agarase. They hydrolyze α -1, 3
linkage and β-1, 4 linkage of agarose respectively
(Duckworth and Turvey, 1969). Agarases are widely used
in food, cosmetic and medical industries for the produc-
tion of oligosaccharides from agar (Kobayashi
et al., 1997; Yoshizawa et al., 1995). Neoagaro-oligo-
saccharides produced by β-agarase inhibit the growth of
bacteria, decrease the degradation rate of starch, and are
used as low-calorie additives to improve food quality
(Ohta et al., 2004). The polysaccharide fractions prepared
from marine algae using β-agarase have macro-
phage-stimulating activity and are usable in physiologi-
cally functional foods with protective and immunopoten-
tiating activities (Yoshizawa et al., 1995). Moreover,
agarases can be used to degrade the cell wall of marine
algae for extraction of labile substances with biological
activities and for preparation of protoplasts (Araki et al.,
1998). In biotechnology, they can also be used to recover
DNA fragments from agarose gel after electrophoresis.
The agaB gene encoding a novel β-agarase AgaB was
cloned from marine Pseudoalteromonas sp. CY24①. It
has been suggested that AgaB may represent a new fam-
ily of glycoside hydrolases. Structurally, AgaB has no

* Corresponding author. Tel: 0086-532-82031680
E-mail: yuwg66@ouc.edu.cn
significant similarity to any known glycoside hydrolases.
Functionally, AgaB hydrolyzes agarose, producing neoa-
garooctaose and neoagarodecaose as the major end prod-
uct, whereas most of β-agarases hydrolyze agarose, pro-
ducing mainly low degree of neoagaro-oligosaccha-
rides. Although AgaB has been produced in pET24a (+)/E.
coli BL21 (DE3) system, only approximately 3 mg of
AgaB could be obtained extracellularly per liter of culture
supernatant①. Thus, there is a strong demand on a rapid,
large-scale production of recombinant AgaB to explore its
potential valuable applications. In this paper, we report
that a significant amount of pure AgaB proteins could be
successfully obtained with one-step purification followed
by refolding of AgaB from E. coli.
2 Materials and Methods
Expression vector pBV220 was kindly provided by
Prof. Fang (Academy of Military Medical Sciences, Bei-
jing, China).
2.1 Construction of Expression Plasmid pBV-AgaB
DNA manipulation and E. coli transformation were
performed following standard procedures (Sambrook
et al., 1989). Gene encoding AgaB was amplified by PCR
from genomic DNA of Pseudoalteromonas sp. CY24

① Ma, C.P., 2005. Molecular cloning and characterization of a
β-agarase belonging to a novel family of glycoside hydrolase
from marine Pseudoalteromonas sp. CY24. (In contribution).

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ZHANG L. et al.: Purification and Refolding of AgaB from Inclusion Body of E. coli
81
as a template, using forward primer (5’-CCG GAA TTC
CAT ATG TTA AAG CGC CAC CAA G-3’) containing
an EcoRI site (bold) and reverse primer (5’-TGC ACT
GCA GCT AGT GGT GGT GGT GGT GGT GTT GGC
AAG TAT AAC CTG-3’) containing a PstI site (bold) and
a hexa-histidine tag (underlined). The PCR product was
digested with EcoRI and PstI and cloned into the EcoRI
and PstI sites of pBV220 vector, yielding the recombinant
plasmid pBV-AgaB. The insert was confirmed by DNA
sequencing.
2.2 Expression of AgaB
Ten microlitres of LB medium containing 50 µg mL-1
ampicillin was inoculated with a single colony of E. coli
BL21 (DE3) transformed with pBV-AgaB and was then
incubated overnight at 37. Fresh LB medium was i
℃
oculated 1:100 with the overnight culture and incubated at
30
until OD
℃
AgaB-His6 was induced by incubation at 42
and cells were harvested by centrifugation (12000 g, 10
min, 4 ℃) and stored at –20 ℃ until use.
n-
600 of about 0.6 was reached. Expression of
for 4
℃
h,
2.3 Isolation and Solubilization of Inclusion Body
The frozen cells were resuspended in 100 mL of lysis
buffer (50 mmol L-1 Tris-HCl, 1mmol L-1 EDTA, and
0.1mol L-1 NaCl, pH 8.0; 100 µg mL-1 lysozyme and 0.1%
Triton X-100), and incubated at room temperature for
15 min. The cells were sonicated in an ice/water bath for
20 cycles of pulsing for 10 s and cooling for 10 s. The
power was set at 30% of the full (750W). The lysate was
centrifugated at 4, 12
℃
taining AgaB inclusion body, was washed twice with
50 mL of wash buffer (0.1 mol L-1 NaCl, 1% TritonX-100,
and 50 mmol L-1 Tris-HCl, pH 8.0). The pellet (about
70 mg) was dissolved and denatured in Buffer A (8 mol L-1
urea, 0.5 mol L-1 NaCl, and 20 mmol L-1 sodium phosphate
(PB), pH 8.0), and incubated at room temperature for
30 min. The insoluble material was removed by centrifu-
gation at 12 000g for 30 min.
000 g for 15 min. The pellet, con-
2.4 Purification of AgaB
A HiTrap Chelating HP column (1mL, Amersham Bio-
sciences) was equilibrated on a FPLC apparatus (Phar-
macia) with 10 bed volumes of Buffer A. After loading
the supernatant of solubilized inclusion body, the column
was washed with the same buffer until the A280 absorbance
reached the baseline. Then the bound protein was eluted
with 50% Buffer B (8 mol L-1 urea, 0.5 mol L-1 NaCl,
20 mmol L-1 PB, pH 4.0). All flow rates were 0.5 mL min-1.
The eluted fractions were monitored with a UV detector
and further analyzed by SDS-PAGE.
2.5 Refolding of AgaB
The purified AgaB was diluted to 0.3 mg mL-1 with
Buffer A. Three milliliters of solution was added to
300 mL of renaturing buffer in three times, one milliliter
each time, and in an interval of 30 min. The solution was
incubated at 4
judged to be complete, the solution was desalted and
concentrated with Amicon Ultra-15 centrifugal filter
(Millipore) by centrifugation at 400 g and 4
The supernatant of the refolded AgaB was collected and
stored at −20℃.
for 24
℃
h with stirring. Once folding was

℃ for 30 min.
2.6 Measurement of AgaB Activity
Ten microlitres of diluted solution of the refolded
AgaB was incubated in 990 µL 20mmol L-1 PB (pH 6.0)
containing 0.25% agarose at 40
matic activity of AgaB was expressed as the initial rate of
agarose hydrolysis by measuring the release of reducing
ends, using the 3, 5-dinitrosalicylic acid procedure (Miller,
1959). One unit of enzymatic activity was defined as the
amount of proteins that produced 1µmol of reducing
sugar per minute under assaying condition. D-galactose
was used as the standard.
for 10min. The enz
℃
y-
2.7 Characterization of the Enzyme
The optimum temperature was determined by monitor-
ing enzyme activity at temperature ranging from 10 to
60
. The enzyme was incubated with substrate solution
℃
(pH6.0) for 1 h at different temperatures. Then the ther-
mostability of the enzyme was evaluated by measuring
the residual activity. The pH dependence of AgaB was
detected at 40 between pH 5.7 and 7.0, an
℃
acidity on the enzyme stability was determined by meas-
uring the residual activity after incubating AgaB at 4
for 1 h at various pHs (pH 4.0-10.6). The effects of vari-
ous metal ions and chelators on enzyme activity were also
examined by determining the residual activity in the
presence of 1 mmol L-1 of the various compounds under
the same condition.
d the effect of

℃
2.8 Hydrolysis Product Analysis of AgaB
Fluorophore assisted carbohydrate electrophoresis (FACE)
was used to identify the hydrolysis products of AgaB.
Enzymatic hydrolysis of agarose was carried out by add-
ing refolded AgaB (60 µg) into 50 mL 20 mmol L-1 PB (pH
6.0) containing 0.25 % agarose at 25 ℃. After incubation
for 24 h, the reaction supernatant was fluorescently la-
beled using monopotassium 7-amino-1, 3- napthalenedi-
sulfonic acid (ANDS) and sodium cyanoborohydride re-
agents (Yu et al., 2002). The degradation products were
subjected to density gradient (18% to 25%) discontinuous
polyacrylamide gel electrophoresis (DGGE) performed on
a vertical slab gel system (Amersham). Six microlitres of
the sample were loaded and subjected to electrophoresis
at constant 300V for 2 h. Finally, the gel was visualized
under UV light and photographed.
2.9 Determination of Protein Concentration
Protein concentration was determined using Bradford
Kit according to the manufacture’s instruction (Beyotime
Institute of Biotechnology, Haimen, China).

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Journal of Ocean University of China Vol.6, No.1, 2007
82

3 Results and Discussion
In this study, the agaB gene was amplified from ge-
nomic DNA of Pseudoalteromonas sp. CY24. As shown
in Fig.1, the agaB gene was inserted into the E. coli ex-
pression vector pBV220. A hexa-histidine (H6) affinity tag
was added to the C-terminus of AgaB to facilitate its puri-
fication.

Fig.1 Map of the plasmid pBV-AgaB for expression
of AgaB-His.
3.1 Expression of Recombinant AgaB

Fig.2 SDS-PAGE analysis of recombinant AgaB.
All samples were analyzed on a 15% gel and
stained with Coomassie Blue. Lane 1, whole cell
lysate of the induced bacteria; Lane 2, supernatant
of whole cell lysate; Lane 3, inclusion bodies; Lane
4, washed inclusion body; Lane 5, protein molecu-
lar weight standards. The bold arrow points to the
band corresponding to AgaB.
In our work, two tandem strong promoters, PR and PL
in pBV220 were used to drive the expression of recombi-
nant AgaB. The promoters were completely repressed at
30℃. The expression of recombinant AgaB-His was in-
duced after the temperature was shifted to 42
pression system controlled by temperature shift is much
more simple and cheaper than that controlled by a chemi-
cal inducer. SDS-PAGE analysis revealed that the recom-
binant AgaB accumulated up to 50% of the total protein
of E. coli cells (Fig.2). Protein in inclusion body is of
great advantage for protein purification, as IB is readily
prepared and contains only a few impurities (Mayer and
Buchner, 2004). After cells being harvested, they were
disrupted by sonication. Inclusion bodies were collected
and washed with 1% TritonX-100 to remove cell debris
and other contaminants (Fig.2).
.
℃ This ex-
3.2 Solubilization and Purification of Inclusion
Bodies
Inclusion body can be solubilized completely by
8 mol L-1 urea. Solubilized IB was not pure enough, which
may associate with other proteins that may interfere with
its refolding. Purification ahead of refolding may be re-
quired to minimize such interference.


Fig.3 (a) SDS-PAGE analysis illustrating the purification
of AgaB after purification using Ni2+ chelating column.
Lane 1, protein molecular weight standards; Lane 2, in-
clusion body dissolved in 8 mol L-1 urea; Lane 3, puri-
fied denatured protein of AgaB by IMAC. (b) Lane 1,
protein molecular weight standards; Lane 2, the super-
natant of refolded protein (concentrated 100-fold from
dilution buffer).
Table 1 Purification of His-tagged AgaB from 1 L
of E. coli BL21 (DE3) cells
Purification step
AgaB-His†
(mg)
Purity††
(%)
Total cell lysate
Denatured inclusion bodies
Nickel chelating chromatography
Refolded protein
Notes: † Yield was determined using a Bradford assay;
†† Purity was estimated from SDS gel.

HisTrap HP is a ready-to-use column, prepacked with
precharged Ni Sepharose. This prepacked column is ideal
for preparative purification of His-tagged recombinant
proteins by immobilized metal ion affinity chromatogra-
85
74
61
44
52.7
85
98
100

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ZHANG L. et al.: Purification and Refolding of AgaB from Inclusion Body of E. coli
83
phy (IMAC). Herein, the inclusion body was solubilized
with Buffer A (8.0 molL-1 urea) and loaded onto the col-
umn and, subsequently, the majority of them bound with
Ni2+-Sepharose. Then the denatured AgaB was eluted
from the column in one-step and analyzed by SDS-PAGE.
As showed in Fig.3a, the purity of approximately 100%
was reached. The results of this purification are summa-
rized in Table 1.
3.3 Refolding of AgaB
Refolding is a process that leads to a change in pro-
tein conformation from unfolded to folded (native) state.
It is initiated by reducing concentration of denaturant
used to solubilize IB (Tsumoto et al., 2003). Optimal
procedure to reduce denaturant concentration and assis-
tance of refolding by solvent additives play key roles in
protein refolding. In general, in particular for refolding
by dilution, low concentration of urea is included in re-
folding solvent (De Bernardez Clark et al., 1998). In our
study, one molar per liter of urea is low for efficient re-
folding, but enough to maintain solubility and flexibility
of folding intermediates. However, urea alone is insuffi-
cient for efficient refolding. The addition of co-solutes is
often essential to facilitate refolding. Without co-solutes,
refolding generates a varying degree of aggregates. As
shown in Table 2, the optimal composition of refolding
buffer for AgaB was 20 mmol L-1 PB, 1 mol L-1 urea,
500 mmol L-1 L-arginine, 10 % glycerol, pH 7.0. In addi-
tion, the optimal temperature of refolding and the initial
concentration of denatured protein were also investi-
gated (Table 2). It was found that 4
were the most appropriate.
Several methods, including dilution, dialysis, diafil-
tration, gel filtration and immobilization onto a solid
support, may be employed to remove or reduce excess
denaturing agents, allowing proteins to renature (Clark,
1998). The simplest and most widely used method for
refolding is dilution. But the method of direct dilution
leads to large volume of solutions and needs large vol-
ume of container, which is impractical for preparation. A
‘pulsed renaturation’ method, whereby the denatured
protein is added to the refolding buffer in pulses (Ru-
dolph and Fischer, 1990), is useful for preparative work
(Middelberg, 2002). In pulsed dilution, after an aliquot
of denatured protein solution is diluted into a refolding
solvent, refolding is allowed to occur for 30 min before
addition of the next aliquot (Tsumoto et al., 2003). In
our experiment, after addition of 3 aliquots of denatured
AgaB to the refolding buffer, the solution was kept at
4
℃ with stirring to avoid high local concentration of
protein, which may result in aggregation. The final pro-
tein concentration was up to 0.03 mg mL-1. In order to
check if the protein folded correctly, the diluted solution
was concentrated by ultra-filtration to remove additives
and the unfolded protein (Fig.3b). The measurement of
activity and protein concentration of the refolded protein
showed that about 40 mg pure enzymes with a high ac-
and 300
℃
µg mL-1
tivity of 1600 units (mg protein)-1 were obtained from 1 L
culture.
Table 2 Establishment of optimal refolding
conditions of denatured AgaB
Parameter
Detergents
Tween 20
Temperature
4 ℃
25 ℃
30 ℃
37 ℃
Protein concentration
300 µg mL-1
>300 µg mL-1
Denaturant
1 mol L-1urea
L-Arginine
500 mmol L-1
Glycine
10%
Notes: RT, room temperature; ‘−’, no effect on refolding;
‘+’, an increased yield of soluble protein.
Effect

−

+
Mild Aggregation
Aggregation
Aggregation

+
Aggregation
+
+++

++
3.4 Enzymatic Properties of Refolded AgaB
The properties of the refolded protein are similar to
those of the native. The optimal temperature and pH of
the refolded protein were 40 ℃ and pH 6.0, respectively.
Refolded AgaB was very stable when pH changed from
5.7 to 10.6. Cu2+, Mn2+ and EDTA were found to inhibit
its activity, whereas Ca2+ and Ba2+ slightly increased its
activity (data not shown). The hydrolyzing product of
refolded AgaB was consistent with that of the native
(Fig.4). It hydrolyzes agarose, producing neoagarooctaose
and neoagarodecaose as the major end products.

Fig.4 FACE analysis of hydrolysis product of agarose
by refolded AgaB. Lane 1, hydrolysis products of aga-
rose by native AgaB; Lane 2, hydrolysis products from
agarose by pure refolded AgaB; Lane 3, neoagarotet-
rose (DP2) and - hexaose (DP3) (Sigma) as the stan-
dard.
Conclusion
We have developed an efficient approach to produce
large amount of AgaB proteins. Even though we did not
scale-up the production, a significant amount of pure

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Journal of Ocean University of China Vol.6, No.1, 2007
84
proteins could be produced cheaply and simply under
moderate fermentation condition. About 40 mg pure en-
zymes with a high activity of 1600 units (mg protein)-1
were obtained from 1 L culture. It is expected that the
production of AgaB on an industrial scale may be real-
ized.
Acknowledgements
This work was supported by a grant from the National
High Technology Research and Development Program of
China (863 Program) (2004AA625020).
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