牛RECQL解旋酶的原核表达纯化及解旋条件优化
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摘要
【目的】通过大肠杆菌表达纯化牛(Bos taurus)RECQL蛋白,利用停流光谱技术对解旋条件进行优化,为研究RECQL的酶促反应动力过程奠定基础。【方法】将人工合成的牛RECQL蛋白编码序列与pET21a载体相连接,获得重组表达载体pET21a-BtRecql后,将其导入大肠杆菌BL21(DE3)菌株中进行诱导表达,经过Ni-NTA亲和层析和Superdex 200凝胶过滤层析,得到纯化的重组蛋白BtRECQL;再利用停流光谱技术对BtRECQL解旋过程进行检测分析,通过摸索不同的试验参数找到BtRECQL发挥解旋功能较适宜的条件。【结果】得到了纯度大于95%的BtRECQL蛋白,产量为0.29 mg/L。在BtRECQL浓度为60nmol/L,反应条件为1.0 mmol/L ATP,30mmol/L Tris-HCl,pH 7.5,60mmol/L NaCl,1mmol/L MgCl2,2mmol/L DTT,孵育温度为37℃时,该蛋白解旋活性较好。【结论】成功表达并纯化了BtRECQL蛋白,确定了其最优的解旋条件。
【Objective】In this study,the RECQL helicase of Bos taurus was expressed and purified in Escherichia coli and the unwinding conditions were optimized by stopped-flow kinetic assays to provide basis for further study of RECQL unwinding activity.【Method】Recombinant plasmid pET21a-BtRecql was constructed by inserting the synthetic RECQL gene of B.taurusinto the expression vector pET21 a.Then,the recombinant plasmid was transformed into E.coli host strain BL21(DE3)and induced to expression.RECQL proteins were purified by Ni-NTA affinity chromatography,followed by gel filtration chromatography(Superdex 200)on FPLC system.Finally,the full-length RECQL was obtained.Through analyzing unwinding process using various stopped-flow assays,the optimum conditions for RECQL to display the strongest unwinding activity were also determined.【Result】The BtRECQL with purity of>95% was obtained and the optimum unwinding conditions were:BtRECQL 60nmol/L,ATP concentration 1mmol/L,reaction temperature 37 ℃,reaction buffer 30 mmol/L Tris-HCl,pH 7.5,60 mmol/L NaCl,1 mmol/L MgCl2,and 2mmol/L DTT.【Conclusion】BtRECQL was expressed and purified successfully and the unwinding conditions were optimized.
【Objective】In this study,the RECQL helicase of Bos taurus was expressed and purified in Escherichia coli and the unwinding conditions were optimized by stopped-flow kinetic assays to provide basis for further study of RECQL unwinding activity.【Method】Recombinant plasmid pET21a-BtRecql was constructed by inserting the synthetic RECQL gene of B.taurusinto the expression vector pET21 a.Then,the recombinant plasmid was transformed into E.coli host strain BL21(DE3)and induced to expression.RECQL proteins were purified by Ni-NTA affinity chromatography,followed by gel filtration chromatography(Superdex 200)on FPLC system.Finally,the full-length RECQL was obtained.Through analyzing unwinding process using various stopped-flow assays,the optimum conditions for RECQL to display the strongest unwinding activity were also determined.【Result】The BtRECQL with purity of>95% was obtained and the optimum unwinding conditions were:BtRECQL 60nmol/L,ATP concentration 1mmol/L,reaction temperature 37 ℃,reaction buffer 30 mmol/L Tris-HCl,pH 7.5,60 mmol/L NaCl,1 mmol/L MgCl2,and 2mmol/L DTT.【Conclusion】BtRECQL was expressed and purified successfully and the unwinding conditions were optimized.
引文
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[2]Brosh R M J.DNA helicases involved in DNA repair and their roles in cancer[J].Nat Rev Cancer,2013,13(8):542-558.
[3]Singleton M R,Dillingham M S,Wigley D B.Structure and mechanism of helicases and nucleic acid translocases[J].Annu Rev Biochem,2007,76:23-50.
[4]Patel S S,Picha K M.Structure and function of hexameric helicases[J].Annu Rev Biochem,2000,69:651-697.
[5]Deng Z,Lehmann K C,Li X,et al.Structural basis for the regulatory function of a complex zinc-binding domain in a replicative arterivirus helicase resembling a nonsense-mediated mRNA decay helicase[J].Nucleic Acids Research,2014,42(5):3464-3477.
[6]Singleton M R,Dillingham M S,Gaudier M,et al.Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks[J].Nature,2004,432(7014):187-193.
[7]Swan M K,Legris V,Tanner A,et al.Structure of human Bloom’s syndrome helicase in complex with ADP and duplex DNA[J].Acta Crystallogr D Biol Crystallogr,2014,70(5):1465-1475.
[8]Manthei K A,Hill M C,Burke J E,et al.Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases[J].Proc Natl Acad Sci,2015,112(14):4292-4297.
[9]Sengoku T,Nureki O,Nakamura A,et al.Structural basis for RNA unwinding by the DEAD-box protein Drosophila Vasa[J].Cell,2006,125(2):287-300.
[10]Cheng Z,Muhlrad D,Lim M K,et al.Structural and functional insights into the human Upf1helicase core[J].EMBO J,2007,26(1):253-264.
[11]Pike A C,Shrestha B,Popuri V,et al.Structure of the human RECQ1helicase reveals a putative strand-separation pin[J].Proc Natl Acad Sci,2009,106(4):1039-1044.
[12]Dillingham M S.SuperfamilyⅠhelicases as modular components of DNA-processing machines[J].Biochem Soc Trans,2011,39(2):413-423.
[13]Croteau D L,Popuri V,Opresko P L,et al.Human RecQ helicases in DNA repair,recombination,and replication[J].Annu Rev Biochem,2014,83:519-552.
[14]Hanada K,Hickson I D.Molecular genetics of RecQ helicase disorders[J].Cell Mol Life Sci,2007,64(17):2306-2322.
[15]Chu W K,Hickson I D.RecQ helicases:multifunctional genome caretakers[J].Nat Rev Cancer,2009,9(9):644-654.
[16]Seki M,Yanagisawa J,Kohda T,et al.Purification of two DNAdependent adenosinetriphosphatases having DNA helicase activity from HeLa cells and comparison of the properties of the two enzymes[J].J Biochem,1994,115(3):523-531.
[17]Puranam K L,Blackshear P J.Cloning and characterization of RECQL,apotential human homologue of the Escherichia coli DNA helicase RecQ[J].J Biol Chem,1994,269(47):29838-29845.
[18]Sharma S,Stumpo D J,Balajee A S,et al.RECQL,a member of the RecQ family of DNA helicases,suppresses chromosomal instability[J].Mol Cell Biol,2007,27(5):1784-1794.
[19]Futami K,Kumagai E,Makino H,et al.Induction of mitotic cell death in cancer cells by small interference RNA suppressing the expression of Recql1helicase[J].Cancer Sci,2008,99(1):71-80.
[20]Sharma S,Brosh R M Jr.Unique and important consequences of RECQ1 deficiency in mammalian cells[J].Cell Cycle,2008,7(8):989-1000.
[21]Popuri V,Huang J,Ramamoorthy M,et al.RECQL5plays cooperative and complementary roles with WRN syndrome helicase[J].Nucleic Acids Res,2013,41(2):881-899.
[22]Lao V V,Welcsh P,Luo Y,et al.Altered RECQ helicase expression in sporadic primary colorectal cancers[J].Transl Oncol,2013,6(4):458-469.
[23]Li D,Frazier M,Evans D B,et al.Single nucleotide polymorphisms of RecQ1,RAD54L,and ATM genes are associated with reduced survival of pancreatic cancer[J].J Clin Oncol,2006,24(11):1720-1728.
[24]Li D,Liu H,Jiao L,et al.Significant effect of homologous recombination DNA repair gene polymorphisms on pancreatic cancer survival[J].Cancer Res,2006,66(6):3323-3330.
[25]Berti M,Chaudhuri A R,Thangavel S,et al.Human RECQ1promotes restart of replication forks reversed by DNA topoisomeraseⅠinhibition[J].Nat Struct Mol Biol,2013,20(3):347-354.
[26]Veith S,Mangerich A.RecQ helicases and PARP1team up in maintaining genome integrity[J].Ageing Res Rev,2015,23:12-28.
[27]Muzzolini L,Beuron F,Patwardhan A,et al.Different quaternary structures of human RECQ1are associated with its dual enzymatic activity[J].PLoS Biol,2007,5(2):e20.
[28]Lucic B,Zhang Y,King O,et al.A prominentβ-hairpin structure in the winged-helix domain of RECQ1is required for DNA unwinding and oligomer formation[J].Nucleic Acids Res,2011,39(5):1703-1717.
[29]Cui S,Klima R,Ochem A,et al.Characterization of the DNAunwinding activity of human RECQ1,a helicase specifically stimulated by human replication protein A[J].J Biol Chem,2003,278(3):1424-1432.
[30]Sami F,Sharma S.Probing genome maintenance functions of human RECQ1[J].Comput Struct Biotechnol,2013,6:e201303014.
[31]Karow J K,Wu L,Hickson I D.RecQ family helicases:roles in cancer and aging[J].Curr Opin Genet Dev,2000,10(1):32-38.
[2]Brosh R M J.DNA helicases involved in DNA repair and their roles in cancer[J].Nat Rev Cancer,2013,13(8):542-558.
[3]Singleton M R,Dillingham M S,Wigley D B.Structure and mechanism of helicases and nucleic acid translocases[J].Annu Rev Biochem,2007,76:23-50.
[4]Patel S S,Picha K M.Structure and function of hexameric helicases[J].Annu Rev Biochem,2000,69:651-697.
[5]Deng Z,Lehmann K C,Li X,et al.Structural basis for the regulatory function of a complex zinc-binding domain in a replicative arterivirus helicase resembling a nonsense-mediated mRNA decay helicase[J].Nucleic Acids Research,2014,42(5):3464-3477.
[6]Singleton M R,Dillingham M S,Gaudier M,et al.Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks[J].Nature,2004,432(7014):187-193.
[7]Swan M K,Legris V,Tanner A,et al.Structure of human Bloom’s syndrome helicase in complex with ADP and duplex DNA[J].Acta Crystallogr D Biol Crystallogr,2014,70(5):1465-1475.
[8]Manthei K A,Hill M C,Burke J E,et al.Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases[J].Proc Natl Acad Sci,2015,112(14):4292-4297.
[9]Sengoku T,Nureki O,Nakamura A,et al.Structural basis for RNA unwinding by the DEAD-box protein Drosophila Vasa[J].Cell,2006,125(2):287-300.
[10]Cheng Z,Muhlrad D,Lim M K,et al.Structural and functional insights into the human Upf1helicase core[J].EMBO J,2007,26(1):253-264.
[11]Pike A C,Shrestha B,Popuri V,et al.Structure of the human RECQ1helicase reveals a putative strand-separation pin[J].Proc Natl Acad Sci,2009,106(4):1039-1044.
[12]Dillingham M S.SuperfamilyⅠhelicases as modular components of DNA-processing machines[J].Biochem Soc Trans,2011,39(2):413-423.
[13]Croteau D L,Popuri V,Opresko P L,et al.Human RecQ helicases in DNA repair,recombination,and replication[J].Annu Rev Biochem,2014,83:519-552.
[14]Hanada K,Hickson I D.Molecular genetics of RecQ helicase disorders[J].Cell Mol Life Sci,2007,64(17):2306-2322.
[15]Chu W K,Hickson I D.RecQ helicases:multifunctional genome caretakers[J].Nat Rev Cancer,2009,9(9):644-654.
[16]Seki M,Yanagisawa J,Kohda T,et al.Purification of two DNAdependent adenosinetriphosphatases having DNA helicase activity from HeLa cells and comparison of the properties of the two enzymes[J].J Biochem,1994,115(3):523-531.
[17]Puranam K L,Blackshear P J.Cloning and characterization of RECQL,apotential human homologue of the Escherichia coli DNA helicase RecQ[J].J Biol Chem,1994,269(47):29838-29845.
[18]Sharma S,Stumpo D J,Balajee A S,et al.RECQL,a member of the RecQ family of DNA helicases,suppresses chromosomal instability[J].Mol Cell Biol,2007,27(5):1784-1794.
[19]Futami K,Kumagai E,Makino H,et al.Induction of mitotic cell death in cancer cells by small interference RNA suppressing the expression of Recql1helicase[J].Cancer Sci,2008,99(1):71-80.
[20]Sharma S,Brosh R M Jr.Unique and important consequences of RECQ1 deficiency in mammalian cells[J].Cell Cycle,2008,7(8):989-1000.
[21]Popuri V,Huang J,Ramamoorthy M,et al.RECQL5plays cooperative and complementary roles with WRN syndrome helicase[J].Nucleic Acids Res,2013,41(2):881-899.
[22]Lao V V,Welcsh P,Luo Y,et al.Altered RECQ helicase expression in sporadic primary colorectal cancers[J].Transl Oncol,2013,6(4):458-469.
[23]Li D,Frazier M,Evans D B,et al.Single nucleotide polymorphisms of RecQ1,RAD54L,and ATM genes are associated with reduced survival of pancreatic cancer[J].J Clin Oncol,2006,24(11):1720-1728.
[24]Li D,Liu H,Jiao L,et al.Significant effect of homologous recombination DNA repair gene polymorphisms on pancreatic cancer survival[J].Cancer Res,2006,66(6):3323-3330.
[25]Berti M,Chaudhuri A R,Thangavel S,et al.Human RECQ1promotes restart of replication forks reversed by DNA topoisomeraseⅠinhibition[J].Nat Struct Mol Biol,2013,20(3):347-354.
[26]Veith S,Mangerich A.RecQ helicases and PARP1team up in maintaining genome integrity[J].Ageing Res Rev,2015,23:12-28.
[27]Muzzolini L,Beuron F,Patwardhan A,et al.Different quaternary structures of human RECQ1are associated with its dual enzymatic activity[J].PLoS Biol,2007,5(2):e20.
[28]Lucic B,Zhang Y,King O,et al.A prominentβ-hairpin structure in the winged-helix domain of RECQ1is required for DNA unwinding and oligomer formation[J].Nucleic Acids Res,2011,39(5):1703-1717.
[29]Cui S,Klima R,Ochem A,et al.Characterization of the DNAunwinding activity of human RECQ1,a helicase specifically stimulated by human replication protein A[J].J Biol Chem,2003,278(3):1424-1432.
[30]Sami F,Sharma S.Probing genome maintenance functions of human RECQ1[J].Comput Struct Biotechnol,2013,6:e201303014.
[31]Karow J K,Wu L,Hickson I D.RecQ family helicases:roles in cancer and aging[J].Curr Opin Genet Dev,2000,10(1):32-38.