Cloning and Expression
of Luffin-b cDNA from the Seeds of Luffa cylindrica
YAN Ming, YANG Xin-Xiu, LI Zhen, PING Bei-Fang, ZHANG
Zu-Chuan*
( Institute of Biochemistry and Cell Biology, Shanghai Institutes for
Biological Sciences, �–hinese Academy of Sciences, Shanghai 200031,
China )
Abstract Luffin-b
with Mr. 28 kD, isolated from the seeds of Luffa cylindrica,is one of
the most toxic single chain plant ribosome inactivating proteins. The cDNA
sequence of luffin-b was already reported by Kataoka in 1992. In this work, the
luffin-b gene(lufB) coding sequence was cloned from the fresh seeds of
Luffa cylindrica by RT-PCR, and the coding sequence of the gene was
shown to be identical with that determined by Kataoka. The lufB
expression plasmid was constructed by inserting the lufB cDNA fragment
into vector pET24a(＋), and the pET24a(＋)-lufB vector was
expressed in E.coli by 0.5 mmol/L IPTG induction. The recombinant
product, which mainly existed in inclusion bodies, was identified by SDS-PAGE
and Western blotting. The recombinant luffin-b, which was renatured by dialysis
with step by step decreasing concentration of urea, showed high activity of
ribosome inactivation.
Key words luffin-b； gene-cloning； expression； Western blot； inclusion body； renature
*Corresponding author： Tel, 86-21-64374430-5281； Fax, 86-21-64338357； e-mail, [email protected]