Cloning and Expression of Luffin-b cDNA from the Seeds of Luffa cylindrica

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.