Expression, purification, crystallization and preliminary crystallographic analysis of the human intracellular chloride channel protein CLIC4.

Abstract

The human chloride intracellular channel protein CLIC4 has been crystallized by the hanging-drop vapour-diffusion technique using trisodium citrate as the precipitant. The best crystals were obtained by the microseeding method. The crystals diffracted to 2.2 A resolution and were found to belong to space group P121, with unit-cell parameters a = 73.19, b =86.05, c = 73.38 A, beta = 112.99 degrees and three molecule per asymmetric unit.

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Protein & Peptide Letters, 2006, 13, 641-643 641
0929-8665/06 $50.00+.00© 2006 Bentham Science Publishers Ltd.
Expression, Purification, Crystallization and Preliminary Crystallographic
Analysis of the Human Intracellular Chloride Channel Protein CLIC4
De-Feng Lia,c,†, Yun-Feng Lia,c,†, Qiu-Hua Huangb, Ying Zhanga and Da-Cheng Wanga,*
aCenter for Structural and Molecular Biology, Institute of Biophysics, Chinese Academy of Science, Beijing, 100101,
People's Republic of China; bShanghai institute of Hematology, Rui Jin Hospital, Shanghai Second Medical Univer-
sity,Shanghai 200025, People’s Republic of China; cGraduate school of Chinese Academy of Sciences, Beijing 100039,
People's Republic of China
Abstract: The human chloride intracellular channel protein CLIC4 has been crystallized by the hanging-drop vapour-
diffusion technique using trisodium citrate as the precipitant. The best crystals were obtained by the microseeding method.
The crystals diffracted to 2.2 Å resolution and were found to belong to space group P121, with unit-cell parameters a =
73.19, b =86.05, c = 73.38 Å, β = 112.99° and three molecule per asymmetric unit.
Keywords: CLIC4, human chloride intracellular channel protein, purification, crystallization, preliminary crystallographic
studies
1. INTRODUCTION
Chloride ion channels are involved in variant functional
performance of the physiological processes. They are located
both within the plasma membrane and other internal cell
membranes [1,2] and participate in the control of secretion
and absorption of salt, regulation of membrane potentials,
organellar acidification, and cell volume homeostasis [3]. To
date, seven members of the CLIC family have been identi-
fied: CLIC1 ,CLIC2,CLIC3, CLIC4, CLIC5, p64, and par-
chorin. Each of these proteins exists both in soluble and in-
tegral membrane forms [4-6]. The chloride intracellular
channel protein CLIC4 is an important member of the CLIC
family and the first human CLIC to be identified recently
[7]. It is expressed in a wide variety of tissues [8-10]. CLIC4
gene expression has been linked to different physiology
functions such as developmental regulation [11], cell differ-
entiation [12] and cell apoptosis [13, 14]. In this paper we
will report the gene cloning, expression, purification, crys-
tallization and preliminary crystallographic analysis of a
soluble form of the intracellular chloride channel protein
CLIC4 from homo sapiens.
2. MATERIALS AND METHODS
2.1. Cloning
The coding sequence for protein CLIC4 (28.7 kDa) was
amplified from RT-PCR products from human haemopoietic
stem cells using the polymerase chain reaction (PCR)
method. The PCR product was purified and restricted with
NdeI and XhoI. The fragment was purified and ligated into
NdeI- and XhoI-restricted sites of the pET22b(+) vector
(Novagen Inc.) with a His6-tag to the C-terminus. The plas-
*Address correspondence to this author at the Center for Structural and
Molecular Biology, Institute of Biophysics, Chinese Academy of Science,
Beijing, 100101, People's Republic of China; Fax: 86-10-64888560; E-mail:
dcwang@sun5.ibp.ac.cn
†The authors contributed equally to this work.
mid was amplified in TG1-competent E. coli cells. This con-
struction was confirmed by DNA sequencing.
2.2. Expression and Purification
The reconmbinant plasmid was transformed into E. coli
host strain BL21 (DE3) and grown on the LB plate contain-
ing ampicillin (100 µg ml-1) and chloramphenicol (30 µg ml-
1) overnight at 310 K. The single clone was picked into 5 ml
LB medium and cultured overnight. Then 1.5 ml culture was
subcultured into 50 ml of fresh LB. Three hours later, 15 ml
culture cells were grown at 310 K in 500 ml LB medium
containing 100 mg ml-1 ampicillin until the OD600 value
reached 0.6. Isopropyl-β-D-thiogalactopyranoside (IPTG)
was added to a final concentration of 1 mM and culture con-
tinued at 310 K for a further 6 h. Cells were collected by
centrifugation at 4000 rev min-1 for 30 min. The pellet was
resuspended in 20 ml lysis buffer (50 mM NaH2PO4 pH 8.0,
300 mM NaCl, 10 mM imidazole) and sonicated. The lysate
was clarified by centrifugation at 16 000 rev min-1 for 10 min
at 277 K to remove the cell debris. The supernatant was ap-
plied to a Ni2+- chelating column [Novagen Inc.] and the
contaminant protein was washed off with wash buffer (50
mM NaH2PO4 pH 8.0, 300 mM NaCl, 20 mM imidazole).
The target protein was eluted with elution buffer (50 mM
NaH2PO4 pH 8.0, 300 mM NaCl, 250 mM imidazole). The
protein was concentrated to 1 ml with an Amicon Ultra 10
kDa molecular weight cutoff filter unit [Millipore] and was
purified on a Superdex-75 column (Pharmacia) in buffer A
(50 mM Tris–HCl pH 8.0).
2.3. Crystallization
The purified protein was concentrated to about 20 mg ml-1
in ultrapure water for crystallization. All crystallization ex-
periments were performed with the hanging-drop vapour-
diffusion method using Hampton Research Index and Crystal
Screens. 1.5 µl protein solution and 1.5 µl reservoir solution
were mixed and equilibrated with 400 µl reservoir solution

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642 Protein & Peptide Letters, 2006, Vol. 13, No. 6Li et al.
in each well at 293 K. The initial microcrystals were ob-
tained in 1.6 M trisodium citrate pH 6.5. The crystallization
condition was optimized at 293 K by varying the concentra-
tion of precipitates (trisodium citrate), pH, and the additives,
but none of the procedures resulted in suitable crystals for X-
ray diffraction. Finally, suitable crystals were obtained by
macro-seeding screening with different equilibration times
and equilibration concentration of precipitates.
2.4. Data Collection and Processing
Diffraction data were collected at Rigaku R-Axis IV++
image plate using with seal-tube X-ray source and Cu K_
radiation (λ =1.5418 Å) from a rotating anode operating at
40 kV and 20 mA with 0.1 mm cofocus incident beam di-
ameter in the National Key Laboratory of Biomacro-
molecules, Institute of Biophysics, CAS. The data were col-
lected at 85 K with a crystal-to-detector distance of 100 mm,
∆φ=1˚ and 300 s exposure time. A total of 180 frames were
collected. Diffraction data were scaled and integrated with
MOSFILM [16]. All data sets were processed by
mosflm6.2.3 program [15] and scaled using CCP4 program
SCALA [16].
3. RESULTS
The gene CLIC4 pET22b(+) vector (Novagen Inc.) with
a his tag was successfully cloned. By transforming the plas-
mid into the E. coli strain BL21(DE3), the recombinant
protein was expressed at a level of about 20 mg in 0.5 L LB
liquid medium.
Hampton Research kit IndexTM and Crystal ScreensTM
were used to supply conditions for initial screening. After
one week the initial microcrystals were obtained in 1.6 M
trisodium citrate pH 6.5. To optimize the growth of the
crystals, the gradient of concentration of precipitates and
equilibration time of reservoir solution were used to macro-
seeding. Finally the best crystals (0.10x2x0.4 mm in size)
(Fig. 1) that qualified for data collection were obtained with
the recipe of 1.5 µl protein solution mixed with 1.5 µl reser-
voir solution which contained 1.1 M trisodium citrate, 0.1 M
Tris pH 7.5 with 0.1 M magnesium chloride and 3% ethanol
as additive, and it was equilibrated for 8 hours in 400 µl res-
ervoir solution before seeding.
Figure 1. Crystals of CLIC4.
The diffraction data were collected at a resolution 2.2 Å
(Fig. 2) and the data statistics are summarized in Table 1.
The results showed that the crystal belongs to space group P
1 21. The unit cell parameters are a =73.18 Å, b =86.05 Å, c
= 73.38 Å, β = 112.99˚. This data are compatible with the
presence of three molecules in the asymmetric unit, with a
Matthews coefficient of 2.50 Å3Da-1 and a solvent content of
51%.
Figure 2. The image of the CLIC4 diffraction pattern of a resolu-
tion 2.2 Å.
Table 1. Crystal data and Data-Collection Statistics. Values
in Parentheses are for the Highest Resolution shell
(2.32-2.20 Å)
Wavelength(Å) 1.5418
Space GroupP21
Unit-cell parameters(Å)
a=73.18,b=86.05,c=73.38,
β(°)=112.99
Molecules per AU3
Matthews coefficient(Å3Da-1 ) 2.5
Resolution range(Å) 23.49-2.20
Unique reflections43497
Completeness (%)99.7(97.7)
Average I/σ (I) 7.8(17.7)
Rsym(%)6.6(36.5)
ACKNOWLEDGEMENTS
This work was supported by the Ministry of Science and
Technology of China and Chinese Academy of Sciences
(KSCXI-SW-17).

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Expression, Purification, Crystallization and Preliminary Crystallographic Analysis Protein & Peptide Letters, 2006, Vol. 13, No. 6 643
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Received: November 29, 2005Revised: February 10, 2006Accepted: February 15, 2006