Biochemical Studies on Superoxide Dismutase from Escherichia coli K12 and Deinococcus radiodurans R1

大腸桿菌與抗輻射奇異球菌中的含錳超強氧歧化酶基因被放大並選殖到表 現載體pQE30 上，使得將要被表現的蛋白質在N 端會帶有His-tag。以IPTG 誘 導並加入cofacotor 錳離子後，重組蛋白可以成功的在E. coli M15 宿主細胞大量 表現。利用Ni-NTA 親和力管柱來進行純化，可以在這一個步驟中得到純度良好 的重組蛋白，之後並利用西方墨點法與質譜分析鑑定所得到的蛋白質。以凝膠層 析法鑑定天然的大腸桿菌含錳超強氧歧化酶、重組的大腸桿菌含錳超強氧歧化 酶、重組的抗輻射奇異球菌含錳超強氧歧化酶，得到它們都是屬於二聚體的蛋白 質。三個蛋白質的比活性分別是13000 U/mg、9800 U/mg、7200 U/mg。 重組的含錳超強氧歧化酶耐熱能力與抗輻射能力都比天然的含錳超強氧歧 化酶要來得好。重組蛋白在攝氏90 度加熱20 分鐘後都還保有70%的活性，而天 然的含錳超強氧歧化酶在攝氏80 度加熱20 分鐘後僅剩20%的活性。重組蛋白接 受2585.88 Gy 的X-ray 照射後，活性都還能保持在80%以上。而天然的蛋白在 相同的劑量下，濃度越低的樣品、活性損失越大。The DNA fragment encoding manganese superoxide dismutase from Deinococcus radiodurans and Escherichia coli was amplified and cloned into the plasmid pQE30 to obtain an N-terminus His-tagged fusion expression plasmid. The recombinant protein MnSODs were successfully overexpressed in E. coli M15 supplemented with Mn2+. The fusion proteins were purified in a single step by Ni-NTA affinity chromatograph, and verified by western blotting and ESI-MASS spectrometry. Characterization of oligomeric state by gel filtration chromatography showed that three kinds of proteins were all dimeric. The specific activity of native E. coli MnSOD, recombinant E. coli MnSOD, and recombinant D. radiodurans MnSOD are 13000 U/mg, 9800 U/mg, and 7200 U/mg respectively. Both recombinant MnSOD is shown to be more thermostable and radiation resistance than native MnSOD of E. coli. The activity of native MnSOD reduced to 20% after heating at 80℃ for 20 min, whereas the activity of both recombinant MnSODs was maintained at about 70% after heating at 90℃ for 20 min. His-tagged MnSOD preserved more than 80% of its activity after irradiation, whereas native MnSOD lost its activity dramatically.中文摘要................................................................................................................................................II ABSTRACT........................................................................................................................................ III LIST OF FIGURES............................................................................................................................ IV LIST OF TABLES ................................................................................................................................V ABBREVIATIONS............................................................................................................................. VI CHAPTER 1. INTRODUCTION..........................................................................................................1 SUPEROXIDE RADICAL ........................................................................................................................2 SUPEROXIDE DISMUTASE ....................................................................................................................3 DEINOCOCCUS RADIODURANS R1........................................................................................................5 CHAPTER 2. MATERIALS AND METHODS...................................................................................7 CHEMICALS (REAGENTS) AND ENZYMES ...........................................................................................8 INSTRUMENTS ....................................................................................................................................9 BACTERIAL STRAINS, AND PLASMIDS..................................................................................................9 TRANSFORMATION...........................................................................................................................10 OVEREXPRESSION AND ENZYME PURIFICATION...............................................................................10 PURIFICATION OF MALTOSE-BINDING PROTEIN (MBP) FUSIONS.....................................................11 PROTEIN CONCENTRATION DETERMINATION...................................................................................12 SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS.......................................12 WESTERN BLOTTING........................................................................................................................12 FAST PROTEIN LIQUID CHROMATOGRAPHY (FPLC)........................................................................13 CRYSTALLIZATION OF E.COLI HIS-TAGGED SOD............................................................................14 ACTIVITY ASSAY OF SUPEROXIDE DISMUTASE AND UNIT DEFINITION..............................................14 TREATMENT OF PROTEINS WITH IONIZING RADIATION...................................................................15 COMPUTATIONAL ANALYSIS..............................................................................................................15 CHAPTER 3. RESULTS AND DISCUSSION...................................................................................16 OVEREXPRESSION AND PURIFICATION OF MNSODS .......................................................................17 THERMOSTABILITY..........................................................................................................................19 RADIORESISTANCE...........................................................................................................................20 REFERENCE:.....................................................................................................................................3