限制性内切酶Mlu I蛋白及其硒代衍生物的制备

Preparation of restriction endonuclease Mlu I and its seleno-derivative

摘要:

【背景】限制性内切酶Mlu I是一种常用的工具酶，在分子生物学领域发挥着重要的作用，其三维结构尚未被解析。【目的】在大肠杆菌中克隆表达、纯化重组Mlu I蛋白及其硒代蛋白，并进行结晶条件的研究。【方法】构建重组表达载体pET28b-Mlu I，在大肠杆菌BL21(DE3)pLysS中诱导表达，利用亲和层析和凝胶过滤层析纯化重组Mlu I蛋白和硒代Mlu I蛋白。对蛋白进行质谱检测、圆二色谱检测以及酶活检测，利用坐滴法进行结晶条件的筛选。【结果】构建了重组表达载体pET28b-Mlu I并纯化获得达到结晶纯度的蛋白，通过质谱检测确定硒代Mlu I蛋白中的8个甲硫氨酸全部被取代，结合酶活测试及圆二色谱检测确定了硒代对Mlu I蛋白的活性、结构无明显影响。采用坐滴法进行初步的晶体生长研究，重组蛋白目前已在1种条件下获得针状晶体并进行初步衍射，获得分辨率在0.32 nm左右的衍射数据。【结论】Mlu I蛋白及硒代Mlu I蛋白纯化体系的构建和结晶条件的研究，可为下一步解析Mlu I三维结构、作用机制的探讨及定向改造奠定基础。

Abstract:

[Background] Restriction endonuclease Mlu I is a commonly used tool in molecular biology. However, its three-dimensional structure has not been determined. [Objective] Mlu I gene was cloned and expressed in Escherichia coli. Mlu I and its seleno-derivative proteins were purified, and the crystallization conditions were studied. [Methods] A recombinant expression vector pET28b-Mlu I was constructed and expressed inductively in E. coli BL21(DE3)pLysS. Affinity and gel filtration chromatography were used to purify Mlu I and Se-Mlu I proteins. Mass spectrometry, circular dichroism and enzyme activity analysis were carried out to characterize these proteins. Sitting drop method was used to screen the crystallization conditions. [Results] The recombinant expression vector pET28b-Mlu I was successfully constructed and the purified Mlu I and Se-Mlu I proteins with purity suitable for crystallization were obtained. All 8 methionines were successfully replaced with se-methionines in the Se-Mlu I protein determined by mass spectrometry. The circular dichroism and enzyme activity analysis confirmed that se-methionines replacement had no significant effect on the activity and structure of the Mlu I protein. Preliminary crystallization study and subsequent X-ray diffraction showed that the needle-like crystal of Mlu I formed under one condition diffracted to a resolution of 0.32 nm. [Conclusion] The construction of Mlu I and Se-Mlu I protein purification system and study of crystallization conditions layed the foundation for further analysis of the three-dimensional structure of Mlu I, revealing the molecular mechanism of Mlu I, and directed evolutionary modification of Mlu I.