牛RECQL解旋酶的原核表达纯化及解旋条件优化

发布时间：2018-11-12 18:23

【摘要】：【目的】通过大肠杆菌表达纯化牛(Bos taurus)RECQL蛋白,利用停流光谱技术对解旋条件进行优化,为研究RECQL的酶促反应动力过程奠定基础。【方法】将人工合成的牛RECQL蛋白编码序列与pET21a载体相连接,获得重组表达载体pET21a-BtRecql后,将其导入大肠杆菌BL21(DE3)菌株中进行诱导表达,经过Ni-NTA亲和层析和Superdex 200凝胶过滤层析,得到纯化的重组蛋白BtRECQL;再利用停流光谱技术对BtRECQL解旋过程进行检测分析,通过摸索不同的试验参数找到BtRECQL发挥解旋功能较适宜的条件。【结果】得到了纯度大于95%的BtRECQL蛋白,产量为0.29 mg/L。在BtRECQL浓度为60nmol/L,反应条件为1.0 mmol/L ATP,30mmol/L Tris-HCl,pH 7.5,60mmol/L NaCl,1mmol/L MgCl2,2mmol/L DTT,孵育温度为37℃时,该蛋白解旋活性较好。【结论】成功表达并纯化了BtRECQL蛋白,确定了其最优的解旋条件。
[Abstract]:[objective] to express and purify bovine (Bos taurus) RECQL protein by Escherichia coli, and to optimize the conditions of unwinding by stop-flow spectroscopy. [methods] the synthetic bovine RECQL protein coding sequence was ligated with the pET21a vector to obtain the recombinant expression vector pET21a-BtRecql. The recombinant protein BtRECQL; was purified by Ni-NTA affinity chromatography and Superdex 200 gel filtration chromatography. Then the BtRECQL unspin process was detected and analyzed by stop-flow spectroscopy. By exploring different test parameters, the suitable conditions for BtRECQL to play its unspin function were found. [results] the purity of BtRECQL protein was more than 95%, and the yield was 0.29 mg/L.. When the concentration of BtRECQL is 60nmol / L and the reaction condition is 1.0 mmol/L ATP,30mmol/L Tris-HCl,pH 7.5 mmol / L NaCl,1mmol/L MgCl2,2mmol/L DTT, the incubation temperature is 37 鈩，

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