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研究生:秦嘉政
研究生(外文):Chia-Cheng Chin
論文名稱:大腸桿菌與抗輻射菌的超氧歧化酶之生化研究
論文名稱(外文):Biochemical Studies on Superoxide Dismutase fromEscherichia coli K12 and Deinococcus radiodurans R1
指導教授:張文章
指導教授(外文):Wen-Chang Chang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:英文
論文頁數:40
中文關鍵詞:大腸桿菌抗輻射菌超氧岐化酶熱穩定性重組蛋白
外文關鍵詞:E. coliDeinococcussuperodxide dismutasethermostabilityrecombinant protein
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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:.....................................................................................................................................38
Amersham International plc (1995). ECL Western Blotting Protocols.
Battista, J.R. 1997. Against all odds: the survival strategies of Deinococcus
radiodurans. Annu Rev Microbiol 51: 203-224.
Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal
Biochem 72: 248-254.
Bull, C., Niederhoffer, E.C., Yoshida, T., and Fee, J.A. 1991. Kinetic Studies of
Superoxide Dismutases: Properties of the Manganese-Containing Protein from
Thermus thermophilus. J Am Chem Soc 113: 4069-4076.
Burnette, W.N. 1981. "Western blotting": electrophoretic transfer of proteins from
sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and
radiographic detection with antibody and radioiodinated protein A. Anal
Biochem 112: 195-203.
Cadenas, E., Boveris, A., Ragan, C.I., and Stoppani, A.O. 1977. Production of
superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase
and ubiquinol-cytochrome c reductase from beef-heart mitochondria. Arch
Biochem Biophys 180: 248-257.
Cooper, J.B., McIntyre, K., Badasso, M.O., Wood, S.P., Zhang, Y., Garbe, T.R., and
Young, D. 1995. X-ray structure analysis of the iron-dependent superoxide
dismutase from Mycobacterium tuberculosis at 2.0 Angstroms resolution
reveals novel dimer-dimer interactions. J Mol Biol 246: 531-544.
Daly, M.J., Ouyang, L., Fuchs, P., and Minton, K.W. 1994. In vivo damage and
recA-dependent repair of plasmid and chromosomal DNA in the
radiation-resistant bacterium Deinococcus radiodurans. J Bacteriol 176:
3508-3517.
Edgington, S.M. 1994. As we live and breathe: free radicals and aging. Correlative
evidence from a number of fields suggests they may be key. Biotechnology (N
Y) 12: 37-40.
Fridovich, I. 1989. Superoxide dismutases. An adaptation to a paramagnetic gas. J
Biol Chem 264: 7761-7764.
Fridovich, I. 1995. Superoxide radical and superoxide dismutases. Annu Rev Biochem
64: 97-112.
Getzoff, E.D., Tainer, J.A., Weiner, P.K., Kollman, P.A., Richardson, J.S., and
Richardson, D.C. 1983. Electrostatic recognition between superoxide and
copper, zinc superoxide dismutase. Nature 306: 287-290.
Halliwell, B., and Gutteridge, J.M.C. 1999. Free radicals in biology and medicine,3rd ed. Oxford University Press, Oxford; New York, pp. xxxi, 936 p.
Hille, R., and Nishino, T. 1995. Flavoprotein structure and mechanism. 4. Xanthine
oxidase and xanthine dehydrogenase. Faseb J 9: 995-1003.
Kitagawa, Y., Tanaka, N., Hata, Y., Kusunoki, M., Lee, G.P., Katsube, Y., Asada, K.,
Aibara, S., and Morita, Y. 1991. Three-dimensional structure of
Cu,Zn-superoxide dismutase from spinach at 2.0 A resolution. J Biochem
(Tokyo) 109: 477-485.
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head
of bacteriophage T4. Nature 227: 680-685.
Markillie, L.M., Varnum, S.M., Hradecky, P., and Wong, K.K. 1999. Targeted
mutagenesis by duplication insertion in the radioresistant bacterium
Deinococcus radiodurans: radiation sensitivities of catalase (katA) and
superoxide dismutase (sodA) mutants. J Bacteriol 181: 666-669.
Martin, M.E., Byers, B.R., Olson, M.O., Salin, M.L., Arceneaux, J.E., and Tolbert, C.
1986. A Streptococcus mutans superoxide dismutase that is active with either
manganese or iron as a cofactor. J Biol Chem 261: 9361-9367.
Matsuura, T., Miyai, K., Trakulnaleamsai, S., Yomo, T., Shima, Y., Miki, S.,
Yamamoto, K., and Urabe, I. 1999. Evolutionary molecular engineering by
random elongation mutagenesis. Nat Biotechnol 17: 58-61.
Mattimore, V., and Battista, J.R. 1996. Radioresistance of Deinococcus radiodurans:
functions necessary to survive ionizing radiation are also necessary to survive
prolonged desiccation. J Bacteriol 178: 633-637.
McCord, J.M., and Fridovich, I. 1969. Superoxide dismutase. An enzymic function
for erythrocuprein (hemocuprein). J Biol Chem 244: 6049-6055.
Minton, K.W., and Daly, M.J. 1995. A model for repair of radiation-induced DNA
double-strand breaks in the extreme radiophile Deinococcus radiodurans.
Bioessays 17: 457-464.
Misra, H., and Fridovich, I. 1976. Superoxide dismutase and the oxygen enhancement
of radiation lethality. Arch Biochem Biophys 176: 577-581.
Nilsson, R., Pick, F.M., and Bray, R.C. 1969. EPR studies on reduction of oxygen to
superoxide by some biochemical systems. Biochim Biophys Acta 192:
145-148.
Parker, M.W., and Blake, C.C. 1988. Crystal structure of manganese superoxide
dismutase from Bacillus stearothermophilus at 2.4 A resolution. J Mol Biol
199: 649-661.
Repine, J.E., Pfenninger, O.W., Talmage, D.W., Berger, E.M., and Pettijohn, D.E.
1981. Dimethyl sulfoxide prevents DNA nicking mediated by ionizing
radiation or iron/hydrogen peroxide-generated hydroxyl radical. Proc Natl Acad Sci U S A 78: 1001-1003.
Rotilio, G., Calabrese, L., Finazzi Agro, A., and Mondovi, B. 1970. Indirect evidence
for the production of superoxide anion radicals by pig kidney diamine oxidase.
Biochim Biophys Acta 198: 618-620.
Sligar, S.G., Lipscomb, J.D., Debrunner, P.G., and Gunsalus, I.C. 1974. Superoxide
anion production by the autoxidation of cytochrome P450cam. Biochem
Biophys Res Commun 61: 290-296.
Steinman, H.M., and Hill, R.L. 1973. Sequence homologies among bacterial and
mitochondrial superoxide dismutases. Proc Natl Acad Sci U S A 70:
3725-3729.
Stohs, S.J., and Bagchi, D. 1995. Oxidative mechanisms in the toxicity of metal ions.
Free Radic Biol Med 18: 321-336.
Tainer, J.A., Getzoff, E.D., Beem, K.M., Richardson, J.S., and Richardson, D.C. 1982.
Determination and analysis of the 2 A-structure of copper, zinc superoxide
dismutase. J Mol Biol 160: 181-217.
Tanaka, A., Hirano, H., Kikuchi, M., Kitayama, S., and Watanabe, H. 1996. Changes
in cellular proteins of Deinococcus radiodurans following gamma-irradiation.
Radiat Environ Biophys 35: 95-99.
Ukeda, H., Maeda, S., Ishii, T., and Sawamura, M. 1997. Spectrophotometric assay
for superoxide dismutase based on tetrazolium salt
3''--1--(phenylamino)-carbonyl--3,
4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate reduction
by xanthine-xanthine oxidase. Anal Biochem 251: 206-209.
Wang, P., and Schellhorn, H.E. 1995. Induction of resistance to hydrogen peroxide
and radiation in Deinococcus radiodurans. Can J Microbiol 41: 170-176.
White, O., Eisen, J.A., Heidelberg, J.F., Hickey, E.K., Peterson, J.D., Dodson, R.J.,
Haft, D.H., Gwinn, M.L., Nelson, W.C., Richardson, D.L., et al. 1999.
Genome sequence of the radioresistant bacterium Deinococcus radiodurans
R1. Science 286: 1571-1577.
Whittaker, M.M., and Whittaker, J.W. 1998. A glutamate bridge is essential for dimer
stability and metal selectivity in manganese superoxide dismutase. J Biol
Chem 273: 22188-22193.
Youn, H.D., Kim, E.J., Roe, J.H., Hah, Y.C., and Kang, S.O. 1996. A novel
nickel-containing superoxide dismutase from Streptomyces spp. Biochem J
318 (Pt 3): 889-896.
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