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| Study of Prokaryotic Expression and Biological Activity of Homo sapiens Kras Protein |
| LI Cui-lin, ZHANG Fan, CHEN Dan-yang, WANG Hao, GUO Qiang, DU Jun |
| Department of Microbial and Biochemical Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China |
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Abstract Objective: To construct a prokaryotic expression plasmid of human TNFα with SUMO,obtain recombinant protein by expression and purification,which is expected to provide basis for the further research and utilization of TNFα.Methods: The gene encoding mature TNFα protein was amplified from plasmid pET32a-hTNFα by PCR and cloned into pET28a with SUMO tag on the upstream.The fusion protein SUMO-hTNFα was expressed in BL21(DE3) engineering bacteria,then was purified by Ni-NTA Resin purification system.The purified production was digested by SUMO protease. One more purification in order to obtain mature TNFα protein.The biological activity was measured in a cytotoxicity assay using L929 cells by CCK-8 test. Results: The prokaryotic expression plasmid pET28a-SUMO-hTNFα was successfully constructed.The results identified by enzyme digestion and nucleotide sequencing corresponded exactly well as expected. The fusion protein SUMO-hTNFα expressed in a soluble form in BL21(DE3).Fusion protein was purified by Ni-NTA Resin purification system. Mature hTNFα protein was obtain after digestion by Sumo protease and purification. ED50 of recombinant hTNFα is 12.8μg/ml. Conclusion: The prokaryotic expression plasmid pET28a-SUMO-hTNFα was successfully constructed. TNFα protein was expressed and obtained by digestion and purification. These established a foundation of further research and utilization of hTNFα.
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Received: 08 April 2014
Published: 25 August 2014
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