Expression of recombinant ribosome inactivating protein MAP30 in E.coli and its biological activity

Objective: To clone and produce ribosome inactivating protein MAP30 from the seeds of Momordica charantia L(bitter melon),and to evaluate the biological activity of the recombinant protein.Methods: The DNA sequence encoding MAP30 was cloned from the fresh seeds of Momordica charantia by PCR,the target DNA fragments were sequenced after T-A cloning.The expression plasmid was constructed by inserting the MAP30 fragment into vector pET30a.MAP30 was expressed in E.coli by addition of IPTG into final concentration of 1.0 mmol/L.The recombinant MAP30 was identified by SDS-PAGE,and the biological activity of MAP30 protein was evaluated by using MTT assay in cancer cells and normal cells following fluid-phase endocytosis.Results: The nucleotide and amino acid sequences of the cloned MAP30 were identical with those of reported MAP30.The solubility of recombinant protein was analyzed by SDS-PAGE,and the MAP30 was mainly produced in soluble form.The recombinant MAP30 showed a greater cytotoxicity to cancer cells than that to normal cells.Conclusion: The gene of MAP30 has been successfully cloned.The recombinant MAP30 protein expressed by E.coli is bioactive.