Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 663-668
doi:10.1111/j.1745-7270.2006.00208x
Expression, Purification and
Anti-tumor Activity of Curcin
Meng-jun Luo1,
Xin-yu Yang1, Wei-Xin Liu2,
Ying Xu1, Ping
Huang2, Fang Yan1,
and Fang Chen1*
1 College
of Life Sciences, Sichuan University, Chengdu 610041, China;
2
Chengdu Institute for Family Planning, Chengdu 610031, China
Received: February
19, 2006
Accepted: June 22,
2006
This work was supported
by the grants from the Tenth Five Years Key Program of the State Science and
Technology Commission in China (2002BA901A and 2004BA411B01)
*Corresponding author: Tel/Fax, 86-28-85417281; E-mail,
Abstract Curcin, purified from the seeds of Jatropha curcas,
can be used as a cell-killing agent. Understanding the anti-tumor activity of
the recombinant protein of curcin is important for its application in
anti-tumor medicine. The segment encoding the mature protein of curcin was
inserted into Escherichia coli strain M15, and the recombinant strain
was induced to express by the optimal revulsant isopropyl-b–D-thiogalactopyranoside at the
concentration of 0.5 mM. The recombinant protein was expressed in the form of
inclusion bodies and purified by Ni-NTA affinity chromatography. The target
protein was incubated with the tumor cells at different concentrations for
different times and the results demonstrated that the target protein could
inhibit the growth of tumor cells (NCL-H446, SGC-7901 and S180) at 5 mg/ml.
Key words curcin; expression; purification; recombinant protein;
anti-tumor activity
Ribosome-inactivating
proteins (RIPs) existing in many plants are N-glycosidases. RIPs can
break the N-glycosidic bond that links the A4324 to the polyphosphate
backbone of the 28S rRNA and thus interrupt protein translation. RIPs are being
studied in the biological and biomedical fields because of their unique
activity as cell-killing agents. They can be classified into three types: type
I RIPs are single-chain with the enzymatic activity and can inhibit cell-free
protein synthesis, but they are relatively non-toxic to cells and animals; type
II and type III RIPs are significantly different from type I RIPs in lectin and
enzymatic activity [1–4]. Curcin, a kind of type I RIP, was first
purified from the seeds of Jatropha curcas by Stirpe et al. 5].
Curcin could inhibit the growth of some tumor cells. In this study we obtained
the sequence encoding the mature protein of curcin by reverse
transcription-polymerase chain reaction (RT-PCR) and expressed it in the
Escherichia coli strain M15. Furthermore, we wanted to find out the purification
and renaturation methods for this recombinant protein and evaluate its in
vitro anti-tumor and anti-virus activity.
Materials and Methods
Materials
The materials
used in this study were obtained from commercial suppliers and used as
received. pQE30 vector, E. coli strain M15, and Ni-NTA agarose column
were purchased from Qiagen (Carlsbad, USA). The RT-PCR kit and DNA clean-up
kit were purchased from TaKaRa (Takara, Japan). The rabbit reticulocyte lysate
system kit was purchased from Promega (Madison, USA). The seeds of J. curcas
were harvested from Panzhihua City (China). The primers were synthesized by
Shanghai Bioengineering Corporation (Shanghai, China). Other reagents and
chemicals were of reagent grade. The cells were grown in LB medium (10 g/L of
tryptone, 5 g/L of yeast extract, 10 g/L of NaCl) unless mentioned otherwise.
DNA sequence and construction
of recombinant strain
Total RNA was
extracted from the seeds of J. curcas by the methods of Zhang et al.
[6]. The primers (forward, 5‘-AACGCATGCGCTGGTTCCACTCCAACTTT-3‘;
reverse, 5‘-ATACTGCAGATACATTGGAAAGATGAGGA-3‘) were designed
according to the sequence of curcin (GenBank accession no. AY069946) [7]. RT-PCR was used to achieve the DNA
sequence encoding the mature protein of curcin. The sequenced segment was
integrated into the pQE30 vector to form pQE30-J1 and expressed in E. coli
strain M15 to form the recombinant strain. A single colony of the recombinant
E. coli strain M15 double-enzyme digested and analyzed by DNA sequencing
was selected for the following experiments.
Optimal induction of
expression conditions of recombination strain
Initial
protein expression screenings were carried out by sodium
dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine if
the colony produced any recombinant protein and where the recombinant protein
existed. The selected colonies were stored at –80 ?C in 20%
(W/V) glycerol. The engineered strain was induced to express the target
protein by different induction conditions. The optimal revulsant reagent was
determined from isopropyl b–D-thiogalactopyranoside (IPTG), xylose
and lactose, and the optimal concentration of revulsant reagent was determined
within 0.01–1.5 mM. The
recombinant protein was induced for different time periods (1–12 h) and
different temperatures as well as adding the optimal revulsant reagent at
different time points to determine the suitable induction time. Different
concentrations (50–400
mg/ml) of
ampicillin were used to find out the most appropriate amount.
SDS-PAGE and Western blot
analysis
SDS-PAGE and Western blot
analysis
Each sample
to be detected by electrophoresis was resuspended in 100 ml ddH2O and mixed with 6?SDS loading
buffer (0.35 M Tris-HCl, pH 6.8, 10% SDS, 36% glycerol, 5% b-mercaptoethanol,
and 0.12% bromophenol blue), and heated at 95 ?C for 8 min. The sample was
centrifuged at 10,000 g for 5 min and 10 ml of supernatant was
analyzed by SDS-PAGE and stained by Coomassie Brilliant Blue R-250. After a
standard PAGE was carried out, western
blot was run as outlined by Sambrook et al. [8]. Western blot was
carried out using anti-RGS-His primary antibody (Huamei, Shanghai, China) and
rabbit anti-mouse secondary antibody (Huamei) conjugated with alkaline
phosphatase.
Purification and refolding of
the recombinant protein
For
overexpression of the recombinant protein, 1 liter of LB medium supplemented
with kanamycin (25 mg/ml)
and ampicillin (100 mg/ml)
was inoculated with 50 ml overnight culture of a single colony of the
recombinant E. coli M15 strain and the mixture was incubated at 28 ?C
with shaking at 250 rpm. IPTG was added to a final concentration of 0.5 mM to
induce the expression of pQE30-J1 at the time point when the absorbance of the
culture at 600 nm reached 0.6–0.8. Then the culture was continuously incubated
for 6 h and the cells were pelleted by centrifugation at 5000 g for 15
min at 4 ?C. The harvested cell paste was resuspended in 50 ml of buffer A (50
mM Tris-HCl, 0.5 mM EDTA, 50 mM NaCl, pH 8.0), lysed with 150 mg/ml lysozyme
and 100 mM phenylmethyl sulfonylfluoride and ultrasonicated. The suspension was
then centrifuged at 10,000 g for 10 min at 4 ?C. The pellet was
resuspended in nine volumes of buffer B (buffer A supplemented with 0.5% Triton
X-100 and 10 mM EDTA) and centrifuged at 10,000 g for 10 min at 4 ?C to
collect the inclusion bodies. The pellet of inclusion bodies was solubilized
with buffers of different pH levels (4–12), denaturants (urea or guanidine-HCl), and a
mixture of denaturant and salt. The suspension was stored at 4 ?C overnight then
centrifuged at 10,000 g for 20 min. The supernatant, after filtering
through a 0.45 mm
filter (Millipore, Bedford, USA), was purified by Ni-NTA agarose affinity
chromatography according to the manual of QIAexpressionist (Qiagen).
The purified
protein (approximately 90% purity) was then refolded by dialysis in
phosphate-buffered saline in a diminishing concentration of urea. Then the
refolded protein was concentrated by lyophilization at –80 ?C.
In vitro activity of the purified
recombinant protein
In this study
we detected the cell-free translation-inhibitory activity of the purified
protein on tumor cells and virus using a Rabbit reticulocyte lysate
system kit (Promega). In brief,
cellular pulmonary cancer NCL-H446, gastric cancer SGC-7901, S180, and Wish cell lines and vesicular stomatitis
virus were resuscitated then incubated with the purified protein at different
concentrations. After 5 d and 7 d, the cultures were examined by flow
cytometer, spectrophotometer and fluoroscopy after staining with acridine
orange. The concentration was designed according to the nature protein.
Treatment at the concentration of zero was considered as the control. The
inhibitory activity of the recombinant protein was estimated by the tests
mentioned above according to Fang and Zhou [9] and Du [10].
Results
Optimal induction conditions
of recombinant protein
It was shown
that the J1 gene segment was correctly inserted into vector pQE30 and suitable
for fusion expression in E. coli strain M15 by double-enzymatic
digestion and DNA sequence analysis for plasmid pQE30-J1. SDS-PAGE and western blot analysis showed that the
engineered strain E. coli M15 could express the target product in the
form of inclusion bodies. Of all the revulsant reagents, and different
concentrations of revulsant reagents, supplied in this study, we found that
IPTG at the concentration of 0.5 mM was optimal for the expression of protein.
other revulsant reagents could barely
induce the expression of the protein within the concentration range of 0.01 mM
to 1.5 mM, and the protein yield was very low. We found that 6 h after the
addition of IPTG at optical density of 0.6 and with the concentration of
ampicillin at 100 mg/ml,
the maximal level of recombinant protein was reached at 28 ?C rather than at 37
?C (data not shown).
Purification of the expressed
protein
In our study,
most of the recombinant protein was expressed in the form of inclusion bodies. Sonication
was suitable to fragment the cells with bacteriolysin in the buffer at 400 W,
99 times at a 5 s interval. To purify the target protein, various buffers with
different pH levels and different denaturants were tried, and we found that the
inclusion bodies could be slightly solubilized with 4 M urea and reach higher
solubilization with 8 M urea at pH 8.0. As Fig. 1 showed, there were two
bands for the purified inclusion body in SDS-PAGE result; and one of the bands
was our target segment as indicated by western
blot. The recombinant protein in phosphate-buffered saline containing 8 M urea
was purified on ana Ni-NTA affinity
column and eluted by 80 mM imidazole.
Activity of the recombinant
protein
Fig. 2 showed that
the refolded recombinant protein showed intense inhibitory activity in the
cell-free translation system at the concentration of 0.1 mg/ml and,
when the concentration was increased to some degree, the inhibitory effect
became higher.
Fig. 3 showed that
the refolded protein could inhibit the growth of several tumor cell lines, such
as cellular pulmonary cancer NCL-H446, gastric cancer SGC-7901 and S180. It had
no effect on Wish cells or the
vesicular stomatitis viruses. It was also shown that, at the concentration of 5
mg/ml, the
protein could inhibit the growth of tumor cells and, when the concentration was
increased to some degree, the inhibitory effect became higher.
Under the
fluorescence microscope, we could find some of the tumor cells (S180) killed by
the recombinant protein compared with the control stained by acridine orange (Fig.
4). It was shown in the former experiment that both the recombinant protein
and the native protein had no effect on the normal cells (data unpublished). We
demonstrated that both of them could inhibit the growth of the tumor cells. It
is also evident from the graphs that the maximal inhibition of the recombinant
protein is a little higher than that of the native protein (Figs. 5–7).
Discussion
Recombinant
plasmid of pQE30-J1 was expressed in E. coli strain M15, and the
purified and refolded protein was tested to determine its toxicity to tumor
cells. IPTG, an efficient lactose manipulator revulsant reagent, is widely used
in many expression studies [11–13]. In our study, the engineered strain could
be more effectively induced to overexpress by IPTG than other two revulsant
reagents. our target product was
overexpressed in inclusion bodies during our experiment. Inclusion body
formation was an enormous problem during protein expression. However, inclusion
bodies can protect short proteins from proteolysis during expression. It is
well known that inclusion bodies occurring during recombinant expression in
bacteria as random protein aggregate in an unfolded, partially folded or
inactive conformational state, can be refolded in vitro to partially
recover their active and native state under defined conditions [14,15].
Denaturants such as urea and guanidine-HCl were added in the buffer to
solubilize the inclusion bodies and in this study urea was better than guanidine-HCl
in solubilization of the recombinant protein. We have found a better way by
using the Ni-NTA affinity column to purify and refold the target protein within
the several methods mentioned above and could only gain 15% renaturation of the
whole harvested protein.
There are
many methods to test the activity of RIPs: (1) quantification of the
inactivation of RIPs by treating with rabbit reticulocyte ribosome [16]; (2)
rapid quantitative determination by HPLC of the chloroacetaldehyde-reactive
material released by RIPs [17]; (3) examination of any bases from 28S rRNA
released by electrophoresis [18]; and (4) direct measurement of the [3H] adenine released by PCR [19]. In our study,
we evaluated the in vitro activity of the recombinant protein using a Rabbit
Reticulocyte Lysate System Kit. Moreover, the selected tumor cells and normal
cells were incubated with the recombinant protein to determine their anti-tumor
activity. In our former study, the recombinant protein could not inhibit the
growth of the normal cells as any other type I RIPs because the recombinant
protein and I RIPs could not infiltrate into the cell (data unpublished).
However, the results suggested that the recombinant protein had significant
influence on the growth of the tumor cells, which was the same as crude curcin
separated from the seeds of J. curcas [20,21]. In the toxicity
experiment we found that our target protein could inhibit the cell-free
translation and kill some tumor cells at a relatively low concentration (0.1 mg/ml and 5 mg/ml,
respectively). Recent studies suggested that RIPs were also capable of inducing
cell death by apoptosis [22,23]. It was also shown in the study that the
morphology of the tumor cells treated with the recombinant protein resembled
cells undergoing death by apoptosis (Fig. 4). Furthermore, we found that
the effect of the recombinant protein on the tumor cells was similar to that of
the native protein on the tumor cells. These results have encouraged us to
continue studying the recombinant protein of curcin in the fields of
immunotoxin and anti-tumor medicine.
References
1 Girbes T, Ferreras JM, Arias FJ, Stirpe F.
Description, distribution, activity and phylogenetic relationship of ribosome-inactivating
proteins in plants, fungi and bacteria. Mini Rev Med Chem 2004, 4: 461–476
2 Nielsen K, Boston RS. Ribosome-inactivating
proteins: A plant perspective. Annu Rev Plant Physiol Plant Mol Biol 2001, 52:
785–816
3 Park SW, Vepachedu R, Sharma N, Vivnco JM.
Ribosome-inactivating proteins in plant biology. Planta 2004, 219: 1093–1096
4 Stirpe F. Ribosome-inactivating proteins.
Toxicon 2004, 44: 371–383
5 Stirpe F, Pession-Brizzi A, Lorenzoni E,
Strocchi P, Montanaro L, Sperti S. Studies on the proteins from the seeds of Croton
tiglium and of Jatropha curcas. Toxic properties and inhibition of
protein synthesis in vitro. Biochem J 1976, 156: 1–6
6 Zhang N, Wei Z, He J, Du L, Liang H. An
efficient and economic method for preparation of high quality plant RNA. Prog
Biochem Biophys 2004, 31: 947–950
7 Lin J, Chen Y, Xu Y, Yan F, Tang
L, Chen F. Cloning and expression of curcin, a ribosome-inactivating protein
from the seeds of jatropha
curcas. Acta Bot Sin 2003, 45: 858–863
8 Sambrook J, Fritsch EF, Maniatis T. Molecular
Cloning: A Laboratory Manual. 2nd ed. New York: Cold Spring Harbor Laboratory
Press 1989
9 Fang F, Zhou L. Modern Medical Experiment
Protocol. Beijing: Chinese Academy Science & Pecking Union Medical College
Press 1995
10 Du P. Medical Experimental Virus. Beijing:
Peoples Military Medical Press 1985
11 Vepachedu R, Park SW, Sharma N, Vivanco JM.
Bacterial expression and enzymatic activity analysis of ME1, a
ribosome-inactivating protein from Mirabilis expansa. Protein Expr Purif
2005, 40: 142–151
12 Goto LS, Beltramini LM, de Moraes DI, Moreira
RA, de Araujo AP. Abrus pulchellus type-2 RIP, pulchellin: heterologous expression and refolding
of the sugar-binding B chain. Protein Expr Purif 2003, 31: 12–18
13 Guo C, Li Z, Shi Y, Xu M, Wise JG, Trommer WE,
Yuan J. Intein-mediated fusion expression, high efficient refolding, and
one-step purification of gelonin toxin. Protein Expr Purif 2004, 37: 361–367
14 Rudolph R, Lilie H. In vitro folding of
inclusion body proteins. FASEB J 1996, 10: 49–56
15 Ceciliani F, Caramori T, Ronchi S, Tedeschi G,
Mortarino M, Galizzi A. Cloning, overexpression, and purification of Escherichia
coli quinolinate synthetase. Protein Expr Purif 2000, 18: 64–70
16 Olsnes S, Fernandez-Puentes C, Carrasco L, Vazquez
D. Ribosome inactivation by the toxic lectins abrin and ricin. Kinetics of the
enzymic activity of the toxin A-chains. Eur J Biochem 1975, 60: 281–288
17 Zamboni M, Brigotti M, Rambelli F, Montanaro
L, Sperti S. High-pressure-liquid-chromatographic and fluorimetric methods for
the determination of adenine released from ribosomes by ricin and gelonin.
Biochem J 1989, 259: 639–643
18 Endo Y, Tsurugi K. RNA N-glycosidase
activity of ricin A-chain. Mechanism of action of the toxic lectin ricin on
eukaryotic ribosomes. J Biol Chem 1987, 262: 8128–8130
19 Brigotti M, Barbieri L, Valbonesi P, Stirpe F,
Montanaro L, Sperti S. A rapid and sensitive method to measure the enzymatic
activity of ribosome-inactivating proteins. Nucleic Acids Res 1998, 26: 4306–4307
20 Lin J, Yan F, Tang L, Chen F. Isolation,
purification and functional investigation in the N-glycosidase activity
of curcin from the seeds of Jatropha curcas. High Technology Letters
2002, 11: 36–40
21 Peumans WJ, Hao Q, Van Damme EJ. Ribosome-inactivating
proteins from plants: more than
RNA N-glycosidases? FASEB J 2001, 15: 1493–1506
22 Narayanan S, Surendranath K, Bora N, Surolia
A, Karande AA. Ribosome inactivating proteins and apoptosis. FEBS Lett 2005,
579: 1324–1331
23 Stirpe F, Barbieri L. Ribosome-inactivating
proteins up to date. FEBS Lett 1986, 195: 1–8