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TECHNICAL NOTE P 138-142
Vapor batch crystallization and preliminary
X-ray crystallographic analysis of a
cold-active endo-β-1,4-glucanase that was
produced through the cold temperature
protein expression
Young Jun An1, Min-Kyu Kim1, Jung Min Song2, Mee Hye Kang2, Youn-Ho Lee2 and Sun-Shin Cha1,3,4*
1
Marine Biotechnology Research Center and 2Marine Ecosystem and Biological Center, Korea Institute of Ocean Science and
Technology, Ansan 426-744, Korea, 3Ocean Science and Technology School, Korea Maritime University, Pusan 606-791, Korea,
4
Department of Marine Biotechnology, University of Science and Technology, Daejeon 305-333, Korea.
*Correspondence: [email protected]
The CaCel gene product from Antarctic springtail Cryptopygus antarcticus (CaCel) belongs to the glycoside hydrolase
family 45 (GH45) type endo-β-1,4-glucanase. Since the production of soluble recombinant CaCel was not successful at the
temperature range of 15-37oC, we further lowered the expression temperature. The Escherichia coli Rosetta-gami2 (DE3)
strain harbouring an expression vector including the CaCel gene was cultured at 10oC. Due to the extremely low growth
rate, the induction time was expanded to 9 days and the 18-liter culture volume was necessary to get enough soluble
protein for crystallization. Crystals of CaCel were grown in droplets under Al’s Oil that allows vapor diffusion. In spite of
small size, the crystal of CaCel, which belonged to the space group P3121, with unit-cell parameters a = 73.57, b = 83.93,
c = 163.77 Å, diffracted to 2.6 Å resolution.
INTRODUCTION
Cellulose, a major component of rigid plant cell walls, is a linear
polysaccharide of glucose residues that are connected to each
other by β-(1→4) linkage. Since this non-food polysaccharide is
the most abundant biomass on earth, the enzymatic degradation
of cellulosic materials is a key technology in biofuel industries.
Endo-glucanases, exo-glucanases, and β-glucosidases are
involved in the breakdown of cellulose. Endo-glucanases
hydrolyze internal bonds of the cellulose chain in a random
way, while exo-glucanases produce cellobiose consisting of
two glucose residues from the chain ends. The conversion of
cellobiose, the major product of the endo- and exo-glucanases,
to glucose is catalyzed by β-glucosidases (Horn et al., 2012).
The freezing-intolerant Antarctic springtail, Cryptopygus
antarcticus Willem (Collembola, Isotomidae), is the most
abundant and widespread terrestrial micro-arthropod in the
maritime Antarctic region. This organism feeds on fungi,
unicellular algae, and detritus (Kim et al., 2014). Therefore, it is
reasonable to search for cold-active enzymes that hydrolyze
polysaccharides in C. antarcticus. An endo-β-1,4-glucanase gene
(CaCel) was identified in the expressed sequence tags (ESTs)
library of C. antarcticus. CaCel is a 225-residue protein with a
putative signal peptide displaying maximum activity at 50 oC
and pH 3.5. Even at 0oC, the cold- adapted CaCel retains more
138 Biodesign l Vol.3 l No.3 l Sep 30, 2015
© 2015 Biodesign
than 40 % activity (Hong et al., 2014). It should be noted that
a cold-adapted cellulase produced by the fungus Acremonium
alcalophilum retained only 20 % activity at 0oC (Hong et al.,
2014).
The GH family 45 is composed of endoglucanses with a
rather small molecular weight (~20 kDa) and broad substrate
specificities for β-1,3/1,4-glucans, such as lichenan and barley
β-glucan, as well as cellulose and its derivatives (Igarashi et
al., 2008). The enzymes in this family catalyze the hydrolysis of
glycosidic bonds via an inversion mechanism that changes the
configuration of the anomeric carbon atom of the -1 glucosyl
group. So far, eight crystal structures of the GH family 45
proteins have been reported, but none of them are cold-active.
CaCel from a psychrotrophic multi-cellular animal belongs to the
GH family 45, and exhibits limited sequence identity (19.72%
~ 55%) to the GH family 45 members whose structures are
available. Therefore, the crystal structure of CaCel will open
new opportunities for structural comparison between fungi and
animal enzymes within the GH family 45 and for the elucidation
of its cold-adaptation mechanism.
One limitation to X-ray crystallographic structural analyses
of proteins is the challenge of preparing milligram quantities
of highly purified soluble proteins. Escherichia coli expression
systems have long been favored as a host organism for
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Young Jun An, Min-Kyu Kim, Jung Min Song, Mee Hye Kang, Youn-Ho Lee and Sun-Shin Cha
structural biologists due to fast growing, low production cost and
convenient manipulation. However, high-level expression of the
target protein in E. coli systems often, unfortunately, led to form
an insoluble complex called inclusion body (Baneyx, 1999). In
such cases, the cultivation temperature of E. coli is sometimes
lowered to suppress the formation of inclusion bodies (Schein,
1989). Generally, protein expression in E. coli system grown at
the sub-optimal temperature (15-30oC) is known to suppress the
formation of inclusion bodies by decelerating the translation of
ribosome, thus to provide a sufficient time for the intermediate to
be folded into its proper structure (Feller et al., 1998). Although
we failed to obtain the soluble form of recombinant CaCel at the
temperature range of 15-37oC, the production of soluble CaCel
was successful at 10 oC that was rarely tried to grow E. coli.
Here, we report the production, crystallization, and preliminary
X-ray crystallographic analyses of CaCel as a first step toward
structure determination.
RESULTS AND DISCUSSION
Expression at 10oC and purification of CaCel
CaCel contains 14 cysteine residues that are supposed to form
seven disulfide bonds according to the program DiANNA 1.1
web server (http://clavius.bc.edu/~clotelab/DiANNA/). Thus, we
selected the E. coli Rosetta-gami2(DE3) strain as an expression
host since the strain is suitable for the expression of proteins
with disulphide bonds (Bessette et al., 1999). According to our
previous report (Song et al., 2012), mesophilic proteins as well
as psychrophilic proteins that were produced as insoluble forms
in E. coli grown at 15-37oC were expressed as soluble forms
when the expression temperature was further lowered to below
A
10oC. In the case of the CaCel production, cell growth reached
the stationary phase and the yield was highest 9 days after IPTG
induction at 10oC (Song et al., 2012). Based on the previous
experiments, we could obtain ~16.2 mg of purified soluble CaCel.
Since the growth rate is drastically decreased at 10oC, we used
18-liter culture volume and a 9-day induction to obtain enough
cell mass. Protein expression at low temperatures is widely
applied to overcome the insolubility problem of recombinant
proteins produced in E.coli. In general, the low-temperature
overexpression of target proteins in E. coli is performed at
the temperature range of 15-25oC. It should be noted that the
production of recombinant proteins at 10oC in E. coli expression
systems is an exceptional case.
We constructed the clone to produce CaCel with a C-terminal
noncleavable His-tag consisting of six histidine residues as
described in methods. To purify soluble CaCel from supernatant
fractions, therefore, we first applied affinity chromatography
using the Ni-NTA agarose resin (Qiagen). The purity of CaCel
eluted by a 300 mM imidazole solution after single washing
step was satisfactory on a SDS-polyacrylamode gel (Figure 1A)
and thus we decided to skip ion exchange chromatography
and hydrophobic interaction chromatography. As a final step to
get proteins for crystallization, size exclusion chromatography
(SEC) was applied. In SEC, proteins are separated depending
on their molecular weights with large proteins eluted ahead
of small proteins. However, CaCel was eluted much later than
expected in the Superdex 75 HR 16/60 size exclusion column
(GE Healthcare): actually, it was eluted at ~72 min (Figure 1A).
According to the calculation of the molecular weight based on
the elution time, the molecular weight of CaCel was estimated
to be 2304 Da, just one tenth of the real molecular weight.
B
FIGURE 1 I Purification. CaCel was purified by affinity chromatography (AC) and size exclusion chromatography (SEC). (A) An elution profile of SEC and a
SDS-PAGE gel showing the purity of CaCel. M: protein size marker, Ni-NTA: CaCel eluted by a 300 mM imidazole solution in AC. SEC (B5): B5 fraction in
SEC, SEC (B4): B4 fraction in SEC. (B) A SDS-PAGE gel showing the purity of the concentrated B4 fraction that was used for crystallization screening.
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139
Vapor batch crystallization of a cold-active cellulase produced at 10oC
A
B
C
FIGURE 2 I Crystallization. Tips for vapor batch crystallization experiments. (A) Processes of vapor batch crystallization setup. (B) A tool for safely picking
up crystals grown under oil. (C) A storage box designed to prevent the fast dehydration of droplets under oil.
Interestingly, we also had observed the same phenomenon in
purifying mannanase from Cryptopygus antarcticus (CaMan)
using SEC (Kim et al., 2013; Kim et al., 2014; Song et al., 2012).
The reason why CaCel and CaMan originated from Antarctic
springtail behaved strange in gel-filtration column remains to be
elucidated.
Vapor batch crystallization
Crystallization experiments of CaCel were performed by vapor
batch crystallization method at 22oC with the concentrated B4
fraction (17 mg/ml) from SEC (Figure 1B). The procedure of our
crystallization setup is as follows. First, the wells of crystallization
plates were filled with Al’s Oil, a 1:1 mixture of Silicon Oil
and Paraffin Oil. Second, 1 μl of crystallization reagents was
pipetted into each well filled with Al’s Oil. To put 1 μl drops
of crystallization reagents on the bottoms of the wells and to
remove bubbles that are formed during pipetting, we centrifuged
crystallization plates immediately after pipetting crystallization
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FIGURE 3 I Needle-shaped crystals. Crystals grew ~22 days after
crystallization setup.
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Young Jun An, Min-Kyu Kim, Jung Min Song, Mee Hye Kang, Youn-Ho Lee and Sun-Shin Cha
reagents into all the wells. Third, 1 μl of protein solution
was pipetted at the droplet of each crystallization reagent.
Finally, Al’s Oil was added to fill the crystallization plates
halfful (Figure 2A and 2B). Since the Al’s Oil is waterpermeable, this kind of batch setup allows vapor diffusion
just like hanging and sitting drop crystallization methods
and thus is called ‘vapor batch crystallization’.
The vapor batch crystallization has the advantage of using
smaller amount of crystallization solutions than the two
conventional vapor diffusion methods. One disadvantage
of the vapor batch crystallization method we experienced
is the fast dry of droplets under Al’s Oil especially in the
dry season. To solve the fast dehydration problem, we
stored crystallization plates on a shelf of a sealed container
with water on the bottom (Figure 2C). To maintain the
moisture of the inside of the container, water is added
and the placement of crystallization plates on the shelf
prevents contact between plates and water. Crystallization
screening was performed with all available screening kits
from Hampton Research and Emerald BioSystems. Initial
crystals (Figure 3) were grown in a precipitant solution of
20% PEG 8K, 0.1 M phosphate-citrate pH 4.2 and 0.2 M
sodium chloride (condition No. 31 of Wizard I from Emerald
BioSystems). The initial crystals were used for data
collection without further optimization.
TABLE 1 I Data collection and processing
Values for the outer shell are given in parentheses.
Diffraction source
17A, Photon Factory
Wavelength (Å)
1.00000
o
Temperature ( C)
- 173
Detector
ADSC Quantum 270 CCD
Crystal-detector distance (mm)
332.8
Rotation range per image (°)
1
Total rotation range (°)
240
Exposure time per image (s)
10
Space group
P3121
a, b, c (Å)
a = b = 81.713, c = 89.352
Mosaicity (°)
0.261
Resolution range (Å)
50.00–2.60 (2.69–2.60)
Total No. of reflections
210251
No. of unique reflections
10971
Completeness (%)
99.5 (99.3)
Redundancy
7.3 (6.4)
I/σ(I)
22.8 (6.8)
+
Rmeas. (%)
12.6
2
Data collection and structure determination
Overall B factor from Wilson plot (Å )
10.8
+
To get diffraction data, crystals under oil must be safely
Rmeas is estimated by multiplying the conventional Rmerge value by the factor [N/(N
− 1)]1/2, where N is the data multiplicity.
picked up without contacting with the oil layer. To pick
up a crystal for mounting, we used a glass capillary that
is connected to a tip through a tube (Figure 2B and 4).
Crystals picked up in this way were transferred to a 1 μl drop
cryoprotectant and mounted by using the cryogenic loop from
of the mother liquor on a coverslip. After incubation about 1~2
Hampton Research (Figure 4). The crystal diffracted to 2.6 Å
seconds, to collect a diffraction data, a crystal was transferred
resolution. The crystals belonged to the primitive hexagonal
to a 1 μl drop of the mother liquor containing 15% glycerol as a
space group, P3121, with unit-cell parameters a = b = 81.713, c =
FIGURE 4 I Crystal mounting. A crystal mounting process using a glass capillary, coverslip, and a cryogenic loop.
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141
Vapor batch crystallization of a cold-active cellulase produced at 10oC
89.352 Å. The crystal volume per unit molecular weight (VM) was
−1
about 1.94 Å3 Da with a solvent content of 36.63% by volume
(Matthews, 1968) when the asymmetric unit was assumed to
contain two molecules (Table 1). Molecular replacement was
performed with the MOLREP program (Vagin and Teplyakov,
2010) using the structure of an endoglucanase (PDB code 1OA7)
that was one of the best matching three dimensional structures
obtained by the sequence-based search of the Protein Data
Bank as a search model. Although we found one solution, we
failed to get the second solution. Analysis of crystal packing
with the found solution clearly showed that there should be two
molecules in the asymmetric unit as expected from the estimated
solvent content. Now we are trying de novo phasing using the
zinc-soaking method (Cha et al., 2012) since we failed to get
crystals of selenomethionine-substituted CaCel.
METHODS
ACKNOWLEDGEMENTS
We thank the staffs at beamlines of BL-5C, Pohang Light Source, Republic
of Korea and BL-17A, Photon Factory, Japan for the data-collection
support. This study was supported by the National Research Foundation
of Korea Grant (NRF-2015R1A2A2A01004168 and NRF-2015M1A5A
1037480) and the KIOST in-house program (PE99314).
AUTHOR INFORMATION The authors declare no potential conflicts of interest.
Original Submission: Aug 25, 2015
Revised Version Received: Sep 10, 2015
Accepted: Sep 11, 2015
REFERENCES
Cloning, expression, purification, and crystallization
The CaCel gene (GeneBank accession No. FJ648735) without the
signal peptide-coding region was amplified by the polymerase chain
reaction using the EST library of C. antarcticus as a template. The entire
CaCel gene product was inserted downstream of the T7 promoter of
the expression plasmid pET-28a (Invitrogen, Carlsbad, CA) using NcoI
and XhoI and the resulting construct expressed residues 17–231 of the
CaCel protein with a C-terminal noncleavable His6 tag (GPHHHHHH).
After verifying the DNA sequence, the plasmid DNA was transformed into
the E. coli Rosetta-gami2(DE3) (Novagen, USA). The cells were grown
to an OD 600 of approximately 0.5 in Luria-Bertani medium containing
50 μg ml -1 Kanamycin (Duchefa), 12.5 μg ml -1 Tetracycline (Duchefa),
50 μg ml -1 Streptomycin(Duchefa) and 34 μg ml -1 chloramphenicol
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and 20 mM imidazole) and an elution buffer (20 mM Tris-HCl pH 7.4 and
300mM imidazole) and Superdex 75 HR 16/60 size exclusion column
(GE Healthcare) with a buffer containing 20 mM Tris pH 7.4 and 1 mM
dithiothreitol (DTT). The CaCel protein was eluted with an apparent
molecular weight in the region of 22 kDa. The purified recombinant CaCel
protein was concentrated to ~17 mg ml-1 for crystallization (Figure 1B).
Crystallization experiments of CaCel protein were performed by the
vapor batch crystallization method at 22oC. Small drops composed of 1
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A crystal was mounted using a nylon loop (50 micron Mounted CryoLoop
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using a Cryostream cooler. Before mounting, crystals were briefly
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142 Biodesign l Vol.3 l No.3 l Sep 30, 2015
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are shown in Table 1.
© 2015 Biodesign
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