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摘要肌红蛋白（Myoglobin，Mb）是一种氧结合血红素蛋白，其主要生理功能是储存氧。Kendrew 教授及其同事 1958 年采用 X-ray 晶体衍射实验报道了首个肌红蛋白三文结构，其成为人们研究蛋白质结构与功能的模型分子。本实验通过基因定点突变技术制备并获得肌红蛋白 H93M 质粒，蛋白在大肠杆菌 BL21(DE3)的感受态细胞中表达成功。利用超声波细胞破碎，经过 65%-95%的硫酸铵分步硫沉，透析后使用 DEAE-sefinose 阴离子交换柱和sephadex G-75凝胶层析分离，并在 DEAE 柱洗脱后添加heme 以防止蛋白纯化过程中 heme 的流失，得到纯度较高的 H93M 突变体蛋白。洗脱过程中用紫外可见分光光度计测其UV/Vis监测蛋白分离情况， 使用SDS-PAGE电泳确定蛋白纯度。 纯化的H93M突变体蛋白与硫化物反应通过紫外可见分子光谱观察，最后用坐滴及悬滴蒸汽扩散法对纯化后的 H93M 突变体蛋白进行结晶，得到针状晶体，并且做 X-衍射对蛋白结构进行分析。41306
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毕业论文关键词 肌红蛋白 H93M 突变体 阴离子交换 凝胶层析 蛋白结晶
Title The purification and structural studies of myoglobin H93Mmutant and its reactions with sulfur containing compounds
Abstract Myoglobin (Mb) is an important muscle protein which plays critical roles in oxygenstorage. Mb was the first protein characterized by X-ray diffraction method andbecame the typical probe to study the structure and function properties of hemecontaining proteins. The vector of H93M mutant was prepared by site-directedmutagenesis and the protein was successfully expressed in E.coli BL21(DE3). Thecell cultures were lysed by ultrasonication. After centrifugation to remove thecell debris, the supernatant was collected for the further purification stepsincluding two steps of ammonium sulfate preipitation. The Mb H93M mutant wasisolated and purified by dialysis, DEAE anion exchange chromatography and G-75gel filtration chromatography. The heme prothetic group was added into the proteinsolution after the IEX procedure. UV-Vis spectroscopy and SDS-PAGE were used tomonitor the purification processes of the H93M protein. The reactions of purifiedprotein with sulfur containing compounds were measured by UV-Vis. Finally, wesuccessfully crystallized Mb H93M protein by using hanging drop and sitting dropvapor diffusion methods.
Keywords myoglobin H93M mutant anion exchange chromatographygel filtration chromatography protein crystallization
目次
1绪论1
1.1肌红蛋白的结构与功能1
1.2肌红蛋白的分离纯化方法2
1.3肌红蛋白与硫化物等反应3
2肌红蛋白突变体H93M的表达、纯化4
2.1实验仪器和材料4
2.2实验方法8
2.2.1肌红蛋白突变体H93M的转化8
2.2.2肌红蛋白的分离纯化9
2.3结果与讨论11
3肌红蛋白突变体H93M的分子光谱及晶体结构解析14
3.1实验仪器和材料14
3.2实验方法15
3.2.1肌红蛋白突变体H93M分子光谱15
3.2.2H93M蛋白结晶16
3.3结果与讨论17
结论21
致谢22
参考文献23
图1.1肌红蛋白结构图1
图2.1H93M蛋白凝胶电泳结果11
图2.2H93N蛋白阴离子交换柱SDS-PAGE电泳胶结果12
图2.3H93M蛋白阴离子交换柱洗脱曲线12
图2.4H93M蛋白heme添加曲线13
图2.5H93M蛋白sephadexG-75柱SDS-PAGE电泳胶结果13
图2.6H93M蛋白葡聚糖凝胶层析柱洗脱曲线14
图3.1Bradford法测定蛋白浓度标准曲线18
图3.2H93M蛋白先后加入Na2S和Na2S2O4反应的紫外分子光谱19
图3.3H93M蛋白先后加入Na2S2O4和Na2S反应的紫外分子光谱19
图3.4160倍下肌红蛋白突变体H93M晶体图20
图3.5肌红蛋白突变体H93MX-衍射结构图20
表2.1实验仪器与设备4
表2.2实验试剂及耗材5
表3.1实验试剂及耗材15
表3.2实验仪器与设备15