期刊文献+
任意字段
题名或关键词
题名
关键词
文摘
作者
第一作者
机构
刊名
分类号
参考文献
作者简介
基金资助
栏目信息

詹氏甲烷球菌DNA连接酶在大肠杆菌中的表达纯化及连接活性鉴定

Expression,Purification and Enzymatic Characterization of a Thermostable DNA Ligase from Methanocaldococcus jannaschii
在线阅读 下载PDF
导出
摘要 对一种耐热性古菌——詹氏甲烷球菌（Methanocaldococcus jannaschii）DNA连接酶进行了克隆、表达、纯化．并对其生物化学特性和酶学活性进行了初步研究．詹氏甲烷球菌DNA连接酶重组蛋白在ATP及Mg^2＋二价阳离子存在的条件下具有连接酶活性，能够封闭DNA链上的切割．通过不同温度下的测试，50-80℃为较适合连接温度，其耐热性强，甚至在90℃下加热5min后仍有连接酶活性；其发挥活性的pH值范围比较宽泛．最适pH值为6．0～9．0．这是国际上对詹氏甲烷球菌DNA连接酶的首次报导． A thermostable DNA ligase gene was identified, and enzymatic properties of the ligase were characterized from Methanocaldococcusjannaschii. It was active when Mg^2＋, Mn^2＋ and Ca^2＋ present as divalent metal cofactor. And assay of thermostability over a range of temperatures showed that the enzyme displayed relative high activity in the range of 50-80 ℃. Furthermore, it also displayed activity at 90℃ for 5 min. The optimal pH range for the ligase was from 6.0 to 9.0. This is the first report of thermostable DNA ligase of Methanocaldococcus jannaschii.
作者 张洋 谢响明 刘建华
机构地区 北京林业大学生物科学与技术学院 上海交通大学生物科学与技术学院
出处 《生命科学研究》 CAS CSCD 2009年第2期99-103,共5页 Life Science Research
关键词 詹氏甲烷球菌 DNA连接酶 蛋白纯化 酶活分析 Methanocaldococcus jannaschii DNA ligase protein purification enzyme activity assay
分类号 Q78 [生物学—分子生物学]
  • 相关文献

参考文献24

  • 1LINDAHL T, BARNES D E. Mammalian DNA ligases [J]. Annu Rev Biochem, 1992, 61(3): 251-281.
  • 2LAI X, SHAO H, HAO F, et al. Biochemical characterization of an ATP-dependent DNA ligase from the hyperthermophilic crenarchaeon Sulfolobus shibatae[J]. Extremophiles, 2002, 6 (6): 469-477.
  • 3ABRAVAYA K,CARRLNO J J. Detection of point mutations with a modified ligase chain reaction (Gap-LCR)[J]. Nucleic Acids Res, 1995,23(3) : 675-682.
  • 4KHANNA M,CAO W G. Ligase detection reaction for identification of low abundance mutations[J]. Clinical Biochemistry, 1999,32(4) : 287-290.
  • 5萨姆布鲁克J 拉塞尔DW 黄培堂译.分子克隆实验指南(第3版)[M].北京:科学出版社,2002..
  • 6MAI V, CHEN X, HONG R, et al. Small abundant DNA binding protein from the thermoacidophilic archaeon Sufolobus shibatae contain negative DNA supercoils[J]. J Bacteriol, 1998, 180(9) : 2560-2563.
  • 7XUE H, GUO R, WEN Y, et ol. Anbundant DNA binding protein from the hyperthermophilic archaeon Sufolobus shibatae affects DNA supereoiling in a temperature-depent fashion[J]. J Baeteriol, 2000,182(14) : 3929-3933.
  • 8LEHMAN I R. DNA ligase: structure, mechanism, and function[J]. Science, 1974,186(4166): 790-797.
  • 9NILSSON M, BARBANY G, ANTSON D O, et al. Enhanced detection and distinction of RNA by enzymatic probe ligation [J]. Nat Biotechnol, 2000, 18 (4) : 791-793.
  • 10NILSSON M, ANTSON D O, BARBANY G, et al. RNA-template DNA ligation for transcript analysis[J]. Nucleic Acids Res, 2001, 29(2): 578-581.

二级参考文献38

  • 1Nilsson M,Barbany G,Antson DO,et al.Enhanced detection and distinction of RNA by enzymatic probe ligation[J].Nat Biotechnol,2000,18:791-793.
  • 2Nilsson M,Antson DO,Barbany G,Landegren U.RNA-templated DNA ligation for transcript analysis[J].Nucleic Acids Res,2001,29:578-581.
  • 3Abravaya K,Carrino JJ,Muldoon S,Lee HH.Detection of point mutations with a modified ligase chain reaction (Gap-LCR)[J].Nucleic Acids Res,1995,23:675-682.
  • 4Yu H,Merchant B,Scheffel C,et al.Automated detection of single nucleotide polymorphism in beta-2 adrenergic receptor gene using LCx(R)[J].Clin Chim Acta,2001,308:17-24.
  • 5Benjamin WH Jr,Smith KR,Waites KB.Ligase chain reaction[J].Methods Mol Biol,2003,226:135-50.
  • 6Osiowy C.Sensitive detection of HBsAg mutants by a gap ligase chain reaction assay[J].J Clin Microbiol,2002,40(7):2566-2571.
  • 7Leon Muinos E,Perez Del Molino ML,et al.Use of ligase chain reaction for the rapid diagnosis of lymph node tuberculosis[J].Scand J Infect Dis,2004,36:724-726.
  • 8Ribeiro FK,Dettoni Vdo V,Peres RL,et al.Evaluation of a commercial test based on ligase chain reaction for direct detection of Mycobacterium tuberculosis in respiratory specimens[J].Rev Soc Bras Med Trop,2004,37:431-435.
  • 9Harden SV,Thomas DC,Benoit N,et al.Real-time gap ligase chain reaction:a rapid semiquantitative assay for detecting p53 mutation at low levels in surgical margins and lymph nodes from resected lung and head and neck tumors[J].Clin Cancer Res,2004,10(7):2379-2385.
  • 10Masasyesva BG,Tong BC,Brock MV,et al.Molecular margin analysis predicts local recurrence after sublobar resection of lung cancer[J].Int J Cancer,2005,113(6):1022-1025.

共引文献77

生命科学研究
2009年 第2期

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部