【摘要】：【目的】通過大腸桿菌表達(dá)純化牛(Bos taurus)RECQL蛋白,利用停流光譜技術(shù)對(duì)解旋條件進(jìn)行優(yōu)化,為研究RECQL的酶促反應(yīng)動(dòng)力過程奠定基礎(chǔ)�！痉椒ā繉⑷斯ず铣傻呐ECQL蛋白編碼序列與pET21a載體相連接,獲得重組表達(dá)載體pET21a-BtRecql后,將其導(dǎo)入大腸桿菌BL21(DE3)菌株中進(jìn)行誘導(dǎo)表達(dá),經(jīng)過Ni-NTA親和層析和Superdex 200凝膠過濾層析,得到純化的重組蛋白BtRECQL;再利用停流光譜技術(shù)對(duì)BtRECQL解旋過程進(jìn)行檢測(cè)分析,通過摸索不同的試驗(yàn)參數(shù)找到BtRECQL發(fā)揮解旋功能較適宜的條件�！窘Y(jié)果】得到了純度大于95%的BtRECQL蛋白,產(chǎn)量為0.29 mg/L。在BtRECQL濃度為60nmol/L,反應(yīng)條件為1.0 mmol/L ATP,30mmol/L Tris-HCl,pH 7.5,60mmol/L NaCl,1mmol/L MgCl2,2mmol/L DTT,孵育溫度為37℃時(shí),該蛋白解旋活性較好。【結(jié)論】成功表達(dá)并純化了BtRECQL蛋白,確定了其最優(yōu)的解旋條件。
[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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