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

【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。

[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 pET28 b-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 pET28 b-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.