核酸外切酶Ⅷ截短体的重组表达及其在体外DNA重组反应中的应用
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摘要
核酸外切酶Ⅷ(ExonucleaseⅧ,ExoⅧ)是一种不依赖于ATP的dsDNA 5?-3?核酸外切酶,可作为体外DNA重组反应极具应用价值的候选蛋白。目前关于ExoⅧ在体外DNA重组反应中的应用尚未有文献报道。本研究构建了保留完整外切活性的截短ExoⅧ(Truncated exonucleaseⅧ,tExoⅧ)的重组表达载体pET28a-tExoⅧ,实现了tExoⅧ在大肠杆菌中的高效表达,在纯化获得高纯度蛋白的基础上,对体外重组反应的温度、反应时间、同源臂长度等因素进行了优化分析。研究结果表明,tExoⅧ在大肠杆菌中以可溶性形式高效表达,每升可纯化92.40 mg tExoⅧ,比活力为1.21×10~5 U/mg;在10μL的重组体系中,2.5 U的tExoⅧ于25℃反应12.5min随后50℃保温50 min时重组效率最高。添加Pfu DNA聚合酶的体外同源臂延伸策略可以有效提高重组克隆的效率。以转化效率为2.2×10~6 CFU/μg的Mach1T1为受体细胞,对于含有21 bp同源臂的1 kb片段与5.8 kb线性化载体的重组,每微克载体可形成1.1×10~4个重组克隆,且阳性率大于80%。同源臂长度在8–21bp范围内,重组反应效率随着同源臂长度增加而提高。在最佳反应条件下,同源臂长度仅为8bp仍可实现有效的重组反应。ExoⅧ介导的体外重组体系具有酶制备方法简单、对DNA的克隆无酶切位点限制及高重组克隆效率等显著优点,是分子生物学领域具有潜在应用价值的高效基因克隆新体系。
Exonuclease Ⅷ(Exo Ⅷ), an ATP-independent dsDNA 5′-3′ exonuclease, is a candidate protein with great application value for in vitro DNA recombination. However, the application of Exo Ⅷ in DNA recombination in vitro has not been reported. In this study, the recombinant expression vector of the truncated Exo Ⅷ(tExo Ⅷ) with the full exonuclease activity was built and used to achieve the overexpression of tExo Ⅷ in Escherichia coli. Based on the purified tExo Ⅷ protein with high-purity, the feasibility of tExo Ⅷ applied in vitro DNA recombination and effects of the reaction temperatures, reaction duration, and homology arm lengths were examined. The results showed that tExo Ⅷ was highly expressed in soluble form in E. coli. One liter of bacterial culture yielded 92.40 mg of purified tExo Ⅷ with the specific activity of 1.21×10~5 U/mg. In a 10 μL recombination system containing 2.5 U tExo Ⅷ, the highest cloning efficiency was achieved in a reaction at 25 °C for 12.5 min and followed by incubation at 50 °C for 50 min. With addition of Pfu DNA polymerase, the homology arm extension strategy can effectively improve the recombination efficiency. Using competent E. coli Mach1 T1 with 2.2×10~6 cfu/μg transformation efficiency as recipient cell, the recombination of a 1 kb fragment with a 21 bp homology arm and a 5.8 kb linearized vector can form about 1.1×10~4 recombinant clones per μg vector, and the positive rates was over 80%. The recombination efficiency was increased with the increasing length of homology arm ranged from 8 to 21 bp. Under the optimal reaction condition, only 8 bp homology arm can still achieve valid DNA recombination. This novel in vitro DNA recombination system mediated by tExo Ⅷ was particularly characterized by its easy preparation, no limitation on restriction sites and high recombination cloning efficiency. All results revealed that the new efficient gene cloning system has potential application in the field of molecular biology.
Exonuclease Ⅷ(Exo Ⅷ), an ATP-independent dsDNA 5′-3′ exonuclease, is a candidate protein with great application value for in vitro DNA recombination. However, the application of Exo Ⅷ in DNA recombination in vitro has not been reported. In this study, the recombinant expression vector of the truncated Exo Ⅷ(tExo Ⅷ) with the full exonuclease activity was built and used to achieve the overexpression of tExo Ⅷ in Escherichia coli. Based on the purified tExo Ⅷ protein with high-purity, the feasibility of tExo Ⅷ applied in vitro DNA recombination and effects of the reaction temperatures, reaction duration, and homology arm lengths were examined. The results showed that tExo Ⅷ was highly expressed in soluble form in E. coli. One liter of bacterial culture yielded 92.40 mg of purified tExo Ⅷ with the specific activity of 1.21×10~5 U/mg. In a 10 μL recombination system containing 2.5 U tExo Ⅷ, the highest cloning efficiency was achieved in a reaction at 25 °C for 12.5 min and followed by incubation at 50 °C for 50 min. With addition of Pfu DNA polymerase, the homology arm extension strategy can effectively improve the recombination efficiency. Using competent E. coli Mach1 T1 with 2.2×10~6 cfu/μg transformation efficiency as recipient cell, the recombination of a 1 kb fragment with a 21 bp homology arm and a 5.8 kb linearized vector can form about 1.1×10~4 recombinant clones per μg vector, and the positive rates was over 80%. The recombination efficiency was increased with the increasing length of homology arm ranged from 8 to 21 bp. Under the optimal reaction condition, only 8 bp homology arm can still achieve valid DNA recombination. This novel in vitro DNA recombination system mediated by tExo Ⅷ was particularly characterized by its easy preparation, no limitation on restriction sites and high recombination cloning efficiency. All results revealed that the new efficient gene cloning system has potential application in the field of molecular biology.
引文
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[12]Kovall R,Matthews BW.Toroidal structure ofλ-exonuclease.Science,1997,277(5333):1824-1827.
[13]Wu TB,Yang YF,Chen W,et al.Noncanonical substrate preference of lambda exonuclease for5?-nonphosphate-ended ds DNA and a mismatch-induced acceleration effect on the enzymatic reaction.Nucleic Acids Res,2018,46(6):3119-3129.
[14]Zhang YW.Single molecule study of the mechanism of homology recombination mediated by RecA[D].Beijing:University of Chinese Academy of Sciences(Institute of Physics CAS),2017(in Chinese).张宇微.RecA蛋白所介导的同源重组机制的研究[D].北京:中国科学院大学(中国科学院物理研究所),2017.
[15]Gonda DK,Radding CM.By searching processively RecA protein pairs DNA molecules that share a limited stretch of homology.Cell,1983,34(2):647-654.
[16]Bell JC,Plank JL,Dombrowski CC,et al.Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA.Nature,2012,491(7423):274-278.
[17]Joseph JW,Kolodner R.ExonucleaseⅧof Escherichia coli.I.Purification and physical properties.J Biol Chem,1983,258(17):10411-10417.
[18]Joseph JW,Kolodner R.ExonucleaseⅧof Escherichia coli.II.Mechanism of action.J Biol Chem,1983,258(17):10418-10424.
[19]Chang HW,Julin DA.Structure and function of the Escherichia coli ExoⅧprotein,a member of the RecB nuclease domain family.J Biol Chem,2001,276(49):46004-46010.
[20]Zhang YM,Buchholz F,Muyrers JPP,et al.A new logic for DNA engineering using recombination in Escherichia coli.Nat Genet,1998,20(2):123-128.
[21]Muyrers JPP,Zhang YM,Buchholz F,et al.RecE/RecT and Redα/Redβinitiate double-stranded break repair by specifically interacting with their respective partners.Genes Dev,2000,14(15):1971-1982.
[22]Chung CT,Niemela SL,Miller RH.One-step preparation of competent Escherichia coli:transformation and storage of bacterial cells in the same solution.Proc Natl Acad Sci USA,1989,86(7):2172-2175.
[23]Halperin SO,Tou CJ,Wong EB,et al.CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window.Nature,2018,560(7717):248-252.
[24]Huang YN,Wu JR,Zheng B,et al.A recombinant expression and characterization of endo-β-N-acetyglucosaminidase from Enterococcus faecalis.Chin J Agric Biotechol,2018,26(4):698-710(in Chinese).黄云娜,吴今人,郑蓓,等.粪肠球菌来源内切-β-N-乙酰氨基葡萄糖苷酶的重组表达及催化特性.农业生物技术学报,2018,26(4):698-710.
[25]Mitsudome T,Xu J,Nagata Y,et al.Expression,purification,and characterization of endo-β-N-acetylglucosaminidase H using baculovirus-mediated silkworm protein expression system.Appl Biochem Biotechnol,2014,172(8):3978-3988.
[26]Ko CY,Lai YL,Liu WY,et al.Arabidopsis ENDO2:its catalytic role and requirement of N-glycosylation for function.J Agric Food Chem,2012,60(20):5169-5179.
[27]Subramanian K,Rutvisuttinunt W,Scott W,et al.The enzymatic basis of processivity in lambda exonuclease.Nucleic Acids Res,2003,31(6):1585-1596.
[2]Liu C,Lin CS.Research Advance on ligation-independent cloning(LIC)method.Genom Appl Biol,2011,30(1):1081-1085(in Chinese).刘超,林陈水.不依赖于连接反应克隆(LIC)的技术进展.基因组学与应用生物学,2011,30(1):1081-1085.
[3]Motohashi K.Evaluation of the efficiency and utility of recombinant enzyme-free seamless DNA cloning methods.Biochem Biophys Rep,2017,9(C):310-315.
[4]Jacobus AP,Gross J.Optimal cloning of PCRfragments by homologous recombination in Escherichia coli.PLoS ONE,2015,10(3):e0119221.
[5]Doyle SA.High-throughput cloning for proteomics research.Methods Mol Biol,2005,310(310):107-113.
[6]Hsiao KC.Exonuclease III induced ligase-free directional subcloning of PCR products.Nucleic Acids Res,1993,21(23):5528-5529.
[7]Henikoff S.Unidirectional digestion with exonuclease III creates targeted breakpoints for DNAsequencing.Gene,1984,28(3):351-359.
[8]Aslanidis C,de Jong PJ.Ligation-independent cloning of PCR products(LIC-PCR).Nucleic Acids Res,1990,18(20):6069-6074.
[9]Islam MN,Lee KW,Yim HS,et al.Optimizing T4DNA polymerase conditions enhances the efficiency of one-step sequence-and ligation-independent cloning.BioTechniques,2017,63(3):125-130.
[10]Li HQ,Lin CS,Zhang WQ.Methods for highthroughput cloning without ligation reaction.Amino Acid Biotic Resour,2013,35(4):43-46(in Chinese).李华琴,林陈水,张文倩.不依赖连接反应的高通量克隆方法.氨基酸和生物资源,2013,35(4):43-46.
[11]Tseng H.DNA cloning without restriction enzyme and ligase.Biotechniques,1999,27(6):1240-1244.
[12]Kovall R,Matthews BW.Toroidal structure ofλ-exonuclease.Science,1997,277(5333):1824-1827.
[13]Wu TB,Yang YF,Chen W,et al.Noncanonical substrate preference of lambda exonuclease for5?-nonphosphate-ended ds DNA and a mismatch-induced acceleration effect on the enzymatic reaction.Nucleic Acids Res,2018,46(6):3119-3129.
[14]Zhang YW.Single molecule study of the mechanism of homology recombination mediated by RecA[D].Beijing:University of Chinese Academy of Sciences(Institute of Physics CAS),2017(in Chinese).张宇微.RecA蛋白所介导的同源重组机制的研究[D].北京:中国科学院大学(中国科学院物理研究所),2017.
[15]Gonda DK,Radding CM.By searching processively RecA protein pairs DNA molecules that share a limited stretch of homology.Cell,1983,34(2):647-654.
[16]Bell JC,Plank JL,Dombrowski CC,et al.Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA.Nature,2012,491(7423):274-278.
[17]Joseph JW,Kolodner R.ExonucleaseⅧof Escherichia coli.I.Purification and physical properties.J Biol Chem,1983,258(17):10411-10417.
[18]Joseph JW,Kolodner R.ExonucleaseⅧof Escherichia coli.II.Mechanism of action.J Biol Chem,1983,258(17):10418-10424.
[19]Chang HW,Julin DA.Structure and function of the Escherichia coli ExoⅧprotein,a member of the RecB nuclease domain family.J Biol Chem,2001,276(49):46004-46010.
[20]Zhang YM,Buchholz F,Muyrers JPP,et al.A new logic for DNA engineering using recombination in Escherichia coli.Nat Genet,1998,20(2):123-128.
[21]Muyrers JPP,Zhang YM,Buchholz F,et al.RecE/RecT and Redα/Redβinitiate double-stranded break repair by specifically interacting with their respective partners.Genes Dev,2000,14(15):1971-1982.
[22]Chung CT,Niemela SL,Miller RH.One-step preparation of competent Escherichia coli:transformation and storage of bacterial cells in the same solution.Proc Natl Acad Sci USA,1989,86(7):2172-2175.
[23]Halperin SO,Tou CJ,Wong EB,et al.CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window.Nature,2018,560(7717):248-252.
[24]Huang YN,Wu JR,Zheng B,et al.A recombinant expression and characterization of endo-β-N-acetyglucosaminidase from Enterococcus faecalis.Chin J Agric Biotechol,2018,26(4):698-710(in Chinese).黄云娜,吴今人,郑蓓,等.粪肠球菌来源内切-β-N-乙酰氨基葡萄糖苷酶的重组表达及催化特性.农业生物技术学报,2018,26(4):698-710.
[25]Mitsudome T,Xu J,Nagata Y,et al.Expression,purification,and characterization of endo-β-N-acetylglucosaminidase H using baculovirus-mediated silkworm protein expression system.Appl Biochem Biotechnol,2014,172(8):3978-3988.
[26]Ko CY,Lai YL,Liu WY,et al.Arabidopsis ENDO2:its catalytic role and requirement of N-glycosylation for function.J Agric Food Chem,2012,60(20):5169-5179.
[27]Subramanian K,Rutvisuttinunt W,Scott W,et al.The enzymatic basis of processivity in lambda exonuclease.Nucleic Acids Res,2003,31(6):1585-1596.