[Cloning and expression of luffin-a gene from the seeds of Luffa cylindrical].
To clone luffin-a cDNA from the seeds of Luffa cylindrical, and to obtain bioactive recombinant luffin-a protein using the expression vector pET-44a (+) in E. coli.



The cDNA sequence encoding luffin-a was cloned from the fresh seeds of Luffa cylindrical by RT-PCR. The target DNA fragments were sequenced after T-A cloning. The luffin-a expression plasmid was constructed by inserting the luffin-a cDNA fragment into vector pET-44a (+). Luffin-a was expressed in E. coli by addition of IPTG into final concentration 1.0 mmol/L. The recombinant luffin-a was identified by SDS-PAGE. The biological activity of luffin-a protein was evaluated by using the MTT assay in HepG2 cells following fluid-phase endocytosis.



In comparison with the reported luffin-a, the homology of nucleotide sequence of the cloned luffin-a gene was 99.73%, while their amino acid sequences were identical. The solubility of recombinant protein was analyzed by SDS-PAGE, and the luffin-a was mainly produced in inclusion bodies. The recombinant luffin-a, renatured by dialysis of the denatured products, showed a similar cytotoxicity to ricin A chain.



The cDNA of luffin-a has been successfully cloned. The recombinant luffin-a protein expressed by E. coli is bioactive.
Xu
Xiao-rong
XR
Department of Biochemistry and Molecular Biology, College of Medicine, Zhejiang University, Hangzhou 310006, China.
Zhan
Jin-biao
JB
Xia
Zheng
Z
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D029776:Luffa / Q000737:chemistry*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D054789:Ribosome Inactivating Proteins, Type 1; D012639:Seeds / Q000737:chemistry*
D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; C063258:luffin-a protein, Luffa cylindrica