Expression of human myeloid differentiation protein-2 in E.coli

    XU Faª²Liang, GU Changª²Guo, HU Chengª²Xiang , LI Lei

    State Key Laboratory of Trauma, Burns and Combined Injury£¬Department 1, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing 400042, China

    ¡¾Abstract¡¿ AIM: To express a fusion protein composed of human myeloid differentiation proteinª²2 (hMDª²2) and Glutathioneª²Sª²Transferase (GST) in E. coli DHª²5¦Á, to analyze the immunogenicity of GST/hMDª²2 and to separate preliminarily the fusion protein. METHODS: The GST/hMDª²2 expression bacteria were engineered and the culture samples were collected at different times after induction at different temperatures by various doses of IPTG (isopropylª²¦Âª²Dª²thiogalaactopyranoside). SDSª²PAGE was used to analyze the expression level and the solubility of GST/hMDª²2, and the Western Blot was performed to examine the immunogenicity of the fusion protein. The preliminarily fusion protein was separated through the elusion of inclusion body, urea denature and grades dialysis. RESULTS: The fusion protein reached its peak expression level (20% in total bacterial protein) after 0.4 mmol/L IPTG was added at 37¡æ for 4 h, and no soluble GST/hMDª²2 was detected in the supernatant of engineering bacterial lysate. GST/hMDª²2 was detected by Western Blot using an antiª²hMDª²2 mAb. GST/hMDª²2 dissolved in denature liquid containing 8 mol/L carbamide and its purity increased to 40% after grades dialysis. CONCLUSION: GST/hMDª²2 can be expressed in E.coli. ......