Prokaryotic Expression And Purification Of Mouse Leptin

Leptin, the product of obese gene, has the faction of suppressing food intake, preventing obesity and regulating energetic metabolism. Obese gene was cloned by zhang and his company in 1994. The role of leptin has expanded from a narrow position in obesity to effects on biological processes, such as diabetes, appetite, thermogenesis, the immune system and reproduction. Recombinant human leptin has the effects on obese, diabetic subjects and patients with congenital leptin deficiency.Mouse obese gene was amplified by PCR and subcloned into prokaryotic expression vector pET-28a(+) to construct the recombinant pET-28a-lep. Transforming pET-28a-lep into E.coli BL21 construct the engineering strain BL21-pET28a-lep. Induced with IPTG at 37'C, BL21-pET28a-lep could highly express fused mouse leptin in the form of inclusion body. After 6h inducement, the maximum amount of the fused protein produced was 39.2% of the total protein. Denaturation and renaturation of inclusion body were performed with 0.5% Sarkosyl. SDS-PAGE indicated the molecular weight of recombinant protein was about 22.5 KD. Purified the fused protein through Ni2+ resin, the concentration of purified protein is 1.086 mg/ml. Western blot showed the fused protein with His tag could react specifically with anti-His antibody.All the research done in the paper provided basic means and methods for further study on structure of leptin fused protein, biological activity and the connection with other protein involved.