GABA_B受体蛋白在大肠杆菌中的表达、纯化及复性

GABA_B Receptor Protein in E.coli:Expression,Purification and Renaturation

在大肠杆菌中培养含有pET28c质粒的BL(DE3),1 mmol.L-1IPTG诱导表达,收集包涵体蛋白,对其进行初步洗涤纯化,然后用8 mol.L-1尿素溶解,在变性条件下(8 mol.L-1尿素)经镍-次氮基三乙酸(Ni-NTA)柱进行亲和层析纯化,得到的电泳纯的蛋白,纯度可达95%以上。采用柱上复性的方法对包涵体蛋白进行复性,依次以含有6,5,4,3,2,1,0 mol.L-1尿素的Refolding buffer缓冲液洗柱,此方法可实现蛋白的同时纯化及复性。

Vector pET28c is transferred into the expression host BL（DE3） in E. coll. The gene was successfully expressed as a fusion protein GABAB with a His-tag at its N terminal. After the recombinant E. coli was induced with 1 mmol · L^-1 IPTG for 5 h, the percentage of the recombinant protein could get to 50% of the total E. coli protein, and 1 mL culture medium could produce 65 μg GABAB protein. GABAB protein was then purified by Ni-NTA column affinity chromatography under denaturing conditions （8 mol ·L^-1 urea）. The GABAB protein was successfully refolded while it was immobilized on Ni-NTA, by washing it using Refolding buffer with urea concentration as follows 6, 4, 3, 2, 1, 0 mol· L^-1.