Klenow(exo-)的表达、活性测定及其在焦磷酸测序中的应用
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摘要
野生的Klenow聚合酶具有3'-5'外切酶活性,会对引物进行切割而造成焦磷酸测序产生错误的延伸信号。为了制备缺失3'外切酶活性的Klenow聚合酶用于焦磷酸测序反应,该研究全合成了外切酶活性缺失突变的Klenow酶基因编码序列,将其插入到表达载体p ET28a(+)中构建重组质粒,并转化到大肠杆菌中,表达外切酶活性缺失的Klenow酶(Klenow(exo-))。通过优化诱导温度、诱导时间与诱导剂浓度,得到了Klenow(exo-)的最佳表达条件为30℃时诱导表达5 h且IPTG浓度为0.1 mmol/L。利用Ni+柱亲和层析法纯化得到了相对分子质量约为67 000的重组Klenow(exo-)酶,并建立了基于生物发光的酶活测定方法,最终将其用于焦磷酸测序反应。结果表明,制备的重组酶具有良好的聚合酶活性但缺失了3'外切酶活性,可成功测定DNA待测模板序列,为焦磷酸测序技术提供了一个有效的关键酶。
The wild Klenow fragment is the large fragment of E. coli DNA polymeraseⅠ,lacking 5'-3' exonuclease activity while having the 5'-3' polymerase activity and the 3'-5' exonuclease activity of DNA polymeraseⅠ. It was widely used in molecular biology experiment. However,the 3'-5' exonuclease activity of the wild Klenow fragment would cut the sequencing primer when used in pyrosequencing,causing the false sequencing signals. In order to prepare the Klenow fragment lacking 3' exonuclease activity for pyrosequencing,the coding gene fragment of D424 A mutant Klenow with deficient exonuclease activity was synthesized,and inserted into the p ET28a( +) expression vector to construct the recombinant expression vector,named p ET28a( +)-Klenow( exo-),which was transformed into E. coli Arctic ExpressTMto express the exonuclease-deficient Klenow fragment( Klenow( exo-)). By optimizing the temperature time of induction,and the inducer concentrations,the optimal expression condition was obtained as cultivating the recombinant strain,inducing with 0. 1 mmol / L IPTG for 5 h at 30 ℃. The recombinant protein with molecular mass of 67 k Da was purified by using Ni+affinity chromatography and eluted by buffer containing 300 mmol / L imidazole. The SDS-PAGE analysis demonstrated that the recombinant protein had a good purity. Then the activity detecting method of Klenow( exo-) based on bioluminescent reaction was established. And the activity of Klenow( exo-) was calculated as 1. 37 U / μL and the specific activity was354. 12 U / mg. This activity detecting method was free of radiolabel and was expected to be used in determination of other polymerase activity. Finally,the Klenow( exo-) was applied in pyrosequencing. The results showed that the Klenow( exo-) had a good polymerase activity and no 3' exonuclease activity,and could be applied in the pyrosequencing to sequence a single-strand DNA template. Our study provides an effective key enzyme for pyrosequencing.
The wild Klenow fragment is the large fragment of E. coli DNA polymeraseⅠ,lacking 5'-3' exonuclease activity while having the 5'-3' polymerase activity and the 3'-5' exonuclease activity of DNA polymeraseⅠ. It was widely used in molecular biology experiment. However,the 3'-5' exonuclease activity of the wild Klenow fragment would cut the sequencing primer when used in pyrosequencing,causing the false sequencing signals. In order to prepare the Klenow fragment lacking 3' exonuclease activity for pyrosequencing,the coding gene fragment of D424 A mutant Klenow with deficient exonuclease activity was synthesized,and inserted into the p ET28a( +) expression vector to construct the recombinant expression vector,named p ET28a( +)-Klenow( exo-),which was transformed into E. coli Arctic ExpressTMto express the exonuclease-deficient Klenow fragment( Klenow( exo-)). By optimizing the temperature time of induction,and the inducer concentrations,the optimal expression condition was obtained as cultivating the recombinant strain,inducing with 0. 1 mmol / L IPTG for 5 h at 30 ℃. The recombinant protein with molecular mass of 67 k Da was purified by using Ni+affinity chromatography and eluted by buffer containing 300 mmol / L imidazole. The SDS-PAGE analysis demonstrated that the recombinant protein had a good purity. Then the activity detecting method of Klenow( exo-) based on bioluminescent reaction was established. And the activity of Klenow( exo-) was calculated as 1. 37 U / μL and the specific activity was354. 12 U / mg. This activity detecting method was free of radiolabel and was expected to be used in determination of other polymerase activity. Finally,the Klenow( exo-) was applied in pyrosequencing. The results showed that the Klenow( exo-) had a good polymerase activity and no 3' exonuclease activity,and could be applied in the pyrosequencing to sequence a single-strand DNA template. Our study provides an effective key enzyme for pyrosequencing.
引文
[1]Brutlag D,Atkinson M R,Setlow P,et al.An active fragment of DNA polymerase produced by proteolytic cleavage[J].Biochem Biophys Res Commun,1969,37(6):982-989.
[2]Klenow H,Henningsen I.Selective elimination of the exonuclease activity of the deoxyribonucleic acid polymerase from Escherichia coli B by limited proteolysis[J].Proc Natl Acad Sci U S A,1970,65(1):168-175.
[3]Jacobsen H,Klenow H,Overgaard-Hansen K.The N-terminal amino-acid sequences of DNA polymerase I from Escherichia coli and of the large and the small fragments obtained by a limited proteolysis[J].Eur J Biochem,1974,45(2):623-627.
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[8]Ghodke P P,Harikrishna S,Pradeepkumar P I.Synthesis and polymerase-mediated bypass studies of the N2-deoxyguanosine DNA damage caused by a lucidin analogue[J].J Org Chem,2015,80(4):2128-2138.
[9]Hofler K,Sarac I,Meier C.Synthesis of C8-N-Arylamine-Modified2'-Deoxyguanosine-5'-Triphosphates and Their Effects on Primer Extension by DNA Polymerases[J].Chembiochem,2015,16(14):2046-2053.
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[12]Huang H,Wu H,Xiao P,et al.Single-nucleotide polymorphism typing based on pyrosequencing chemistry and acryl-modified glass chip[J].Electrophoresis,2009,30(6):991-998.
[13]Kobayashi N,Shinagawa S,Nagata T,et al.Development of Biomarkers Based on DNA Methylation in the NCAPH2/LMF2Promoter Region for Diagnosis of Alzheimer's Disease and Amnesic Mild Cognitive Impairment[J].PLo S One,2016,11(1):e0146449,1-12.
[14]Leeuwerke M,Eilander M S,Pruis M G,et al.DNA Methylation and Expression Patterns of Selected Genes in First Trimester Placental Tissue from Pregnancies with Small for Gestational Age Infants at Birth[J].Biol Reprod,2016,94(2):37,1-7.
[15]Teschler S,Gotthardt J,Dammann G,et al.Aberrant DNA Methylation of r DNA and PRIMA1 in Borderline Personality Disorder[J].Intern J Molec Sci,2016,17(1):67,1-11.
[16]刘夕群,朱术会,邹秉杰,等.三酶焦测序体系的建立及其在唐氏综合征快速诊断中的应用[J].遗传,2010,32(5):517-523.
[17]Ye H,Wu H,Huang H,et al.Prenatal diagnosis of trisomy 21 by quantitatively pyrosequencing heterozygotes using amniotic fluid as starting material of PCR[J].Analyst,2013,138(8):2443-2448.
[18]张晓丹,武海萍,陈之遥,等.碱基序列标记法结合焦磷酸测序测定不同来源基因表达量[J].分析化学研究报告,2009,37(8):1107-1112.
[19]Song Q,Wu H,Feng F,et al.Pyrosequencing on nicked ds DNA generated by nicking endonucleases[J].Anal Chem,2010,82(5):2074-2081.
[20]Choudhury M A,Lott W B,Banu S,et al.Nature and Extent of Genetic Diversity of Dengue Viruses Determined by 454 Pyrosequencing[J].PLo S One,2015,10(11):e0142473,1-15.
[21]Illeghems K,De Vuyst L,Weckx S.Comparative genome analysis of the candidate functional starter culture strains Lactobacillus fermentum 222 and Lactobacillus plantarum 80 for controlled cocoa bean fermentation processes[J].BMC Genomics,2015,16(1):766,1-13.
[22]Xu M J,Wang J H,Bu X L,et al.Deciphering the streamlined genome of Streptomyces xiamenensis 318 as the producer of the anti-fibrotic drug candidate xiamenmycin[J].Sci Rep,2016,6:18977,1-11.
[23]Ronaghi M,Uhlen M,Nyren P.A sequencing method based on real-time pyrophosphate[J].Science,1998,281(5375):363-365.
[24]Agah A,Aghajan M,Mashayekhi F,et al.A multi-enzyme model for Pyrosequencing[J].Nucleic Acids Res,2004,32(21):e166,1-15.
[25]Derbyshire V,Freemont P S,Sanderson M R,et al.Genetic and crystallographic studies of the 3',5'-exonucleolytic site of DNA polymerase I[J].Science,1988,240(4849):199-201.
[26]Kukreti P,Singh K,Ketkar A,et al.Identification of a new motif required for the 3'-5'exonuclease activity of Escherichia coli DNA polymerase I(Klenow fragment):the RRRY motif is necessary for the binding of single-stranded DNA substrate and the template strand of the mismatched duplex[J].J Biol Chem,2008,283(26):17979-17990.
[27]Setlow P.DNA polymerase I from Escherichia coli[J].Methods Enzymol,1974,29:3-12.
[28]Joyce C M,Kelley W S,Grindley N D.Nucleotide sequence of the Escherichia coli pol A gene and primary structure of DNA polymerase I[J].J Biol Chem,1982,257(4):1958-1964.
[29]罗娟,吴文娟,邹秉杰,等.酿酒酵母ATP-硫酸化酶在大肠杆菌中的表达纯化及其在焦测序中的应用[J].生物工程学报,2007,23(4):623-627.
[30]邹秉杰,罗娟,武海萍,等.生物素化ATP硫酸化酶的表达、固定化与应用[J].生物化学与生物物理进展,2009,36(7):923-928.
[31]沈毅珺,姚见儿,易进华,等.重组大肠杆菌高密度培养的进展[J].药物生物技术,2000,7(4):243-247.
[32]戎晶晶,刁振宇,周国华.大肠杆菌表达系统的研究进展[J].药物生物技术,2005,12(6):416-420.
[2]Klenow H,Henningsen I.Selective elimination of the exonuclease activity of the deoxyribonucleic acid polymerase from Escherichia coli B by limited proteolysis[J].Proc Natl Acad Sci U S A,1970,65(1):168-175.
[3]Jacobsen H,Klenow H,Overgaard-Hansen K.The N-terminal amino-acid sequences of DNA polymerase I from Escherichia coli and of the large and the small fragments obtained by a limited proteolysis[J].Eur J Biochem,1974,45(2):623-627.
[4]Que B G,Downey K M,So A G.Mechanism of selective inhibition of 3'to 5'exonuclease activity of Escherichia coli DNA polymerase I by nucleoside 5'-monophosphates[J].Biochemistry,1978,17(9):1603-1606.
[5]Ollis D L,Brick P,Hamlin R,et al.Structure of large fragment of Escherichia coli DNA polymerase I complexed with d TMP[J].Nature,1985,313(6005):762-766.
[6]Yin X,Tan K,Vajta G,et al.Massively parallel sequencing for chromosomal abnormality testing in trophectoderm cells of human blastocysts[J].Biol Reprod,2013,88(3):69,1-6.
[7]Smith L,Underhill P,Pritchard C,et al.Single primer amplification(SPA)of c DNA for microarray expression analysis[J].Nucleic Acids Res,2003,31(3):e9,1-7.
[8]Ghodke P P,Harikrishna S,Pradeepkumar P I.Synthesis and polymerase-mediated bypass studies of the N2-deoxyguanosine DNA damage caused by a lucidin analogue[J].J Org Chem,2015,80(4):2128-2138.
[9]Hofler K,Sarac I,Meier C.Synthesis of C8-N-Arylamine-Modified2'-Deoxyguanosine-5'-Triphosphates and Their Effects on Primer Extension by DNA Polymerases[J].Chembiochem,2015,16(14):2046-2053.
[10]Wu H,Wu W,Chen Z,et al.Highly sensitive pyrosequencing based on the capture of free adenosine 5'phosphosulfate with adenosine triphosphate sulfurylase[J].Anal Chem,2011,83(9):3600-3605.
[11]Hanami T,Oyama R,Itoh M,et al.New pyrosequencing method to analyze the function of the Klenow fragment(EXO-)for unnatural nucleic acids:pyrophosphorolysis and incorporation efficiency[J].Nucleosides Nucleotides Nucleic Acids,2012,31(8):608-615.
[12]Huang H,Wu H,Xiao P,et al.Single-nucleotide polymorphism typing based on pyrosequencing chemistry and acryl-modified glass chip[J].Electrophoresis,2009,30(6):991-998.
[13]Kobayashi N,Shinagawa S,Nagata T,et al.Development of Biomarkers Based on DNA Methylation in the NCAPH2/LMF2Promoter Region for Diagnosis of Alzheimer's Disease and Amnesic Mild Cognitive Impairment[J].PLo S One,2016,11(1):e0146449,1-12.
[14]Leeuwerke M,Eilander M S,Pruis M G,et al.DNA Methylation and Expression Patterns of Selected Genes in First Trimester Placental Tissue from Pregnancies with Small for Gestational Age Infants at Birth[J].Biol Reprod,2016,94(2):37,1-7.
[15]Teschler S,Gotthardt J,Dammann G,et al.Aberrant DNA Methylation of r DNA and PRIMA1 in Borderline Personality Disorder[J].Intern J Molec Sci,2016,17(1):67,1-11.
[16]刘夕群,朱术会,邹秉杰,等.三酶焦测序体系的建立及其在唐氏综合征快速诊断中的应用[J].遗传,2010,32(5):517-523.
[17]Ye H,Wu H,Huang H,et al.Prenatal diagnosis of trisomy 21 by quantitatively pyrosequencing heterozygotes using amniotic fluid as starting material of PCR[J].Analyst,2013,138(8):2443-2448.
[18]张晓丹,武海萍,陈之遥,等.碱基序列标记法结合焦磷酸测序测定不同来源基因表达量[J].分析化学研究报告,2009,37(8):1107-1112.
[19]Song Q,Wu H,Feng F,et al.Pyrosequencing on nicked ds DNA generated by nicking endonucleases[J].Anal Chem,2010,82(5):2074-2081.
[20]Choudhury M A,Lott W B,Banu S,et al.Nature and Extent of Genetic Diversity of Dengue Viruses Determined by 454 Pyrosequencing[J].PLo S One,2015,10(11):e0142473,1-15.
[21]Illeghems K,De Vuyst L,Weckx S.Comparative genome analysis of the candidate functional starter culture strains Lactobacillus fermentum 222 and Lactobacillus plantarum 80 for controlled cocoa bean fermentation processes[J].BMC Genomics,2015,16(1):766,1-13.
[22]Xu M J,Wang J H,Bu X L,et al.Deciphering the streamlined genome of Streptomyces xiamenensis 318 as the producer of the anti-fibrotic drug candidate xiamenmycin[J].Sci Rep,2016,6:18977,1-11.
[23]Ronaghi M,Uhlen M,Nyren P.A sequencing method based on real-time pyrophosphate[J].Science,1998,281(5375):363-365.
[24]Agah A,Aghajan M,Mashayekhi F,et al.A multi-enzyme model for Pyrosequencing[J].Nucleic Acids Res,2004,32(21):e166,1-15.
[25]Derbyshire V,Freemont P S,Sanderson M R,et al.Genetic and crystallographic studies of the 3',5'-exonucleolytic site of DNA polymerase I[J].Science,1988,240(4849):199-201.
[26]Kukreti P,Singh K,Ketkar A,et al.Identification of a new motif required for the 3'-5'exonuclease activity of Escherichia coli DNA polymerase I(Klenow fragment):the RRRY motif is necessary for the binding of single-stranded DNA substrate and the template strand of the mismatched duplex[J].J Biol Chem,2008,283(26):17979-17990.
[27]Setlow P.DNA polymerase I from Escherichia coli[J].Methods Enzymol,1974,29:3-12.
[28]Joyce C M,Kelley W S,Grindley N D.Nucleotide sequence of the Escherichia coli pol A gene and primary structure of DNA polymerase I[J].J Biol Chem,1982,257(4):1958-1964.
[29]罗娟,吴文娟,邹秉杰,等.酿酒酵母ATP-硫酸化酶在大肠杆菌中的表达纯化及其在焦测序中的应用[J].生物工程学报,2007,23(4):623-627.
[30]邹秉杰,罗娟,武海萍,等.生物素化ATP硫酸化酶的表达、固定化与应用[J].生物化学与生物物理进展,2009,36(7):923-928.
[31]沈毅珺,姚见儿,易进华,等.重组大肠杆菌高密度培养的进展[J].药物生物技术,2000,7(4):243-247.
[32]戎晶晶,刁振宇,周国华.大肠杆菌表达系统的研究进展[J].药物生物技术,2005,12(6):416-420.