Cloning And Expression Of Luffin-a Gene In Escherichia Coli

[Objective]Luffins, a group of ribosome inactivating proteins (RIPs), are able to inactivate eukaryotic protein synthesis by attacking the 28S ribosomal RNA. At present, some types of luffins including luffin-a, luffin-b, luffin-s, luffincylin and luffin-P1 have been isolated and purified. Among them, luffin-a is the most toxic. Recent studies have shown that some RIPs possess strong anti-human immunodeficiency virus (HIV) activity. AU et al examined some plant RIPs including luffins for the ability to interfere with HIV-1 replication in a variety of mechanistic assays in vitro. The results suggested that the anti-HIV property of RIPs may be due to inhibition of HIV-1 integrase. In this article, we report that luffin-a gene has been successfully cloned from the seed of Luffa cylindrical and expressed in E. coli. [Methods]The cDNA sequence encoding luffin-a was cloned from the fresh seeds of Luffa cylindrical by RT-PCR. Nde I site was introduced at the upstream primer whereas a stop codon and Bam H I site were added at the downstream. The nucleotide sequences of the target DNA amplification fragment were sequenced after T-A cloning. The fragments digested with restriction endonucleases Nde I and Bam H I were subsequently cloned into the vector pET-44a(+). E. coli BL21 (DE3) transformed with the expression plasmid was grown in LB at 37℃ to an optical density of 0.6.Expression was induced by adding IPTG to a final concentration of 1mmol/L and incubation with shaking at 37℃ for 3~5 h. The expression product was identified by 12% SDS-PAGE. The result showed that the recombinant product mainly existed in inclusion body. The recombinant protein in inclusion body was denatured with 8mol/L urea and was renatured by dialysis. The renatured proteins were affinitively purified on a Blue-Sepharose 6B column by elution with 1.0 mol/L sodium chloride. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out to check the purity. The cytotoxicity of luffin-a was evaluated by the MTT assay in HepG2 and Hela cells following fluid-phase endocytosis. [Results]In comparison with the reported luffin-a sequence, the nucleotide sequence homology of the cloned luffin-a gene was 99.73 %, while their putative amino acid sequences were identical. The plasmid DNA of pET-44a(+)-lufA extracted from a single colony was digested with Nde I and BamH I. The map of agarose electrophoresis indicated that the constructed plasmid had two bands with 5 600 bp and 760 bp, which proved the target fragment had been inserted. The expression product was identified by SDS-PAGE, and it mainly existed in inclusion bodies. The renatured and purified proteins showed a single band on 12 % SDS-PAGE. The results of MTT assay showed the luffin-a possessed a similar cytotoxicity to RTA. [Conclusion]The expression system of luffin-a has been successfully established. The inclusive bodies can be renatured by dialysis and the renatured luffin-a can bepurified by one-step affinity chromatography on the column of Blue-Sepharose 6B. The purity of final product was over 90 % analyzed by SDS-PAGE. The renatured protein shows cytotoxitic, which suggests that luffin-a can enter the cell byendocytosis.The cDNA of luffin-a was successfully cloned. Luffin-a can be further modified by gene engineering. The recombinant luffin-a was bioactive and may be useful in treatment of AIDS patients.