Li-li Zhang1, Qian Ding, Jin-biao Zhan. 1. Department of Biochemistry and Genetics, College of Medicine, Zhejiang University, Hangzhou 310058, China.
Abstract
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. RESULT: 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.
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. RESULT: 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.