人类髓样分化蛋白-2的原核表达

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

目的 :在大肠杆菌DH 5α中表达人类髓样分化蛋白 2 (humanmyeloiddifferentiatonprotein 2 ,hMD 2 )与谷胱甘肽巯基转移酶 (glutathione s transferase ,GST)的融合蛋白 (GST/hMD 2 ) ,并对融合蛋白进行免疫学鉴定及初步的变性复性处理 .方法 :建立GST/hMD 2表达菌 ,在不同温度下以不同浓度IPTG诱导不同时间后采样 ,用SDS PAGE分析各种组合条件下融合蛋白的表达量和溶解性 ;用Western Blot鉴定融合蛋白免疫性 ;用尿素变性和透析法对融合蛋白进行初步纯化 .结果 :在 37℃经 0 .4mmol/LIPTG诱导 4h后 ,GST/hMD 2可获最高表达量 (约占菌体总蛋白的 2 0 % ) ,未见可溶性表达 ;Western Blot证实GST/hMD 2能与抗MD 2单克隆抗体特异性结合 ;GST/hMD 2溶于 8mol/L尿素 ,梯度透析后纯度提高至4 0 % .结论 :hMD 2可在大肠杆菌DH

AIM: To express a fusion protein composed of human myeloid differentiation protein 2 (hMD 2) and Glutathione S Transferase (GST) in E. coli DH 5α, to analyze the immunogenicity of GST/hMD 2 and to separate preliminarily the fusion protein. METHODS: The GST/hMD 2 expression bacteria were engineered and the culture samples were collected at different times after induction at different temperatures by various doses of IPTG (isopropyl β D thiogalaactopyranoside). SDS PAGE was used to analyze the expression level and the solubility of GST/hMD 2, 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℃ for 4 h, and no soluble GST/hMD 2 was detected in the supernatant of engineering bacterial lysate. GST/hMD 2 was detected by Western Blot using an anti hMD 2 mAb. GST/hMD 2 dissolved in denature liquid containing 8 mol/L carbamide and its purity increased to 40% after grades dialysis. CONCLUSION: GST/hMD 2 can be expressed in E.coli .