| 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60 nmol/L，反应条件为1.0 mmol/L ATP，30 mmol/L Tris-HCl，pH 7.5，60 mmol/L NaCl，1 mmol/L MgCl2，2 mmol/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。 |
| 关键词: 牛 RECQL解旋酶 原核表达纯化 解旋活性 |
| DOI： |
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| 基金项目:国家自然科学基金项目（31370798） |
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| Prokaryotic expression,purification and optimization of unwinding conditions of Bos taurus RECQL helicase |
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ZHAI Liutao,QIN Wei,LIU Nanü,et al
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| Abstract: |
| 【Objective】 In this study, the RECQL helicase of Bos taurus was expressed and purified in Escherichia coli and the unwinding conditions were optimized by stopped-flow kinetic assays to provide basis for further study of RECQL unwinding activity. 【Method】 Recombinant plasmid pET21a-BtRecql was constructed by inserting the synthetic RECQL gene of B.taurus into the expression vector pET21a.Then,the recombinant plasmid was transformed into E.coli host strain BL21(DE3) and induced to expression.RECQL proteins were purified by Ni-NTA affinity chromatography,followed by gel filtration chromatography (Superdex 200) on FPLC system.Finally,the full length RECQL was obtained.Through analyzing unwinding process using various stopped flow assays,the optimum conditions for RECQL to display the strongest unwinding activity were also determined. 【Result】 The BtRECQL with purity of >95% was obtained and the optimum unwinding conditions were:BtRECQL 60 nmol/L,ATP concentration 1 mmol/L,reaction temperature 37 ℃,reaction buffer 30 mmol/L Tris-HCl, pH 7.5,60 mmol/L NaCl,1 mmol/L MgCl2,and 2 mmol/L DTT.【Conclusion】 BtRECQL was expressed and purified successfully and the unwinding conditions were optimized. |
| Key words: Bos taurus RECQL helicase prokaryotic expression and purification DNA unwinding activity |
