ISSN 1021-4437, Russian Journal of Plant Physiology, 2007, Vol. 54, No. 2, pp. 202–206. © Pleiades Publishing, Ltd., 2007.
Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 2, pp. 229–234.
Ribosome-Inactivating Proteins (RIPs) existing in
many plants are N-glycosidases. RIPs can break the
N-glycosidic bond linking the A4324 to the polyphos-
phate backbone of the 28S rRNA of the rat liver ribo-
some 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
comprise a single chain with the enzymatic activity and
can inhibit cell-free protein synthesis but are relatively
nontoxic 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 RIPs,
which was ﬁrst isolated from seeds of
by Stripe  and could inhibit the growth of some
The objective of this study was to obtain the
sequence encoding the mature curcin by RT-PCR and
express it in the
strain M15. We also plan to
develop the methods for puriﬁcation and renaturing of
the recombination protein and to assess its in vitro anti-
MATERIALS AND METHODS
The materials were obtained from com-
mercial suppliers. The strains, pQE30 and M15, and
His-tag column were purchased from Qiagen (Ger-
many). RT-PCR Kit and DNA clean up Kit were pur-
chased from TaKaRa (Japan). The Rabbit Reticulocyte
Lysate System Kit was purchased from Promega
(United States). The seeds of
vested from Panzhihua City, Sichuan Province (China).
The primers were synthesized by Shanghai Bioengi-
neering Co. Other reagents and chemicals were of
reagent grade. The strains used in this study were
grown in LB medium unless mentioned otherwise.
Obtaining of DNA sequence and construction of the
The RNA was extracted from
seeds by the methods of Zhang Nianhui
. The primers were designed according to the
sequence of curcin (GenBank no. AY069946) .
RT-PCR was used to produce the DNA sequence
encoding the mature curcin. Then the fragment was
integrated into the pQE30 vector and expressed in
strain M15 to form the recombinant strain.
A single colony of the recombinant
which was subjected to a double enzymatic digest
DNA-sequence analysis, was selected to do in the fol-
The optimum conditions for the induction of the
recombination strain expression.
Initial protein expres-
sion screening was performed by SDS-PAGE to deter-
mine if the colony produced any recombinant protein
Cloning, Expression, and Antitumor Activity
of Recombinant Protein of Curcin
M. J. Luo
, W. X. Liu
, X. Y. Yang
, Y. Xu
, F. Yan
, P. Huang
, and F. Chen
College of Life and Science, Sichuan University 24, South Section 1, Yihuan Road, Chengdu, Sichuan, 610041 China;
fax: 28-854172571; e-mail: email@example.com
Chengdu Institute for Family Planning, Chengdu, 610031 China
Received March 3, 2006
—Curcin, a protein isolated from the seeds of
can be used as a cell-killing agent. To
elaborate the puriﬁcation methods and investigate the antitumor activity of the recombinant protein, the frag-
ment encoding the mature protein of curcin was inserted into
strain M15 and the recombinant strain was
induced to express by the optimum inducer (0.5 mM isopropyl-
-D-thiogalactopyranoside). The recombinant
protein was expressed in the form of the inclusion body and was puriﬁed by Ni-NTA afﬁnity chromatography.
The protein of interest was incubated with the tumor cells at various concentrations for different time. It was
shown that the target protein could inhibit the growth of NCL-H446, SGC-7901, and S180 at a very low con-
- Esherichia coli - curcin - expression - puriﬁcation - recombinant protein - anti-
: Gn—guanidine chloride; IPTG—isopropyl-
thiogalactopyranoside; PBS—phosphate-buffered saline; PMSF—
phenylmethylsulfonyl ﬂuoride; RIP—ribosome-inactivating pro-
tein; RT-PCR—reverse transcription–polymerase chain reaction.
The text was submitted by the authors in English.