沙门氏菌硫修饰基因dptC的克隆表达与分离纯化
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摘要
【目的】细菌DNA磷硫酰化修饰是指DNA骨架非磷氧桥上的一个氧被硫取代,该修饰增加了机体的抗氧化作用,其发生受被称为dnd的基因簇控制。沙门氏菌(Salmonella entericaserovar Cerro 87)是具有磷硫酰化修饰现象的细菌之一,其dnd基因簇被命名为dptBCDE。本研究旨在克隆其中的dptC基因,优化dptC表达条件,为进一步研究DptC在DNA磷硫酰化修饰过程中的酶学功能奠定基础。【方法】以沙门氏菌总DNA为模板,设计特异引物、PCR扩增获得dptC基因片段,连接于表达载体pGEX-6P-1的SmaI和XhoI位点之间,构建融合表达载体pJTU3622;将pJTU3622转化大肠杆菌(Escherichia coliDH10B),经氨苄霉素抗性初选及序列测定,获得阳性克隆;提取阳性中pJTU3622再转化大肠杆菌表达宿主[E.coli BL21(DE3)pLysS],获得工程菌株Anxh103;优化表达条件,诱导表达dptC基因;采用GST-Trap柱和kata FPLC纯化系统分离纯化DptC蛋白。【结果】获得沙门氏菌dptC基因表达载体pJTU3622和工程菌株Anxh103;确定dptC最佳诱导表达条件为:诱导温度18℃,诱导时间8-18 h,IPTG诱导浓度0.6 mmol/L,LB培养基中添加50μmol/L Fe2+。【结论】成功克隆了沙门氏菌dptC基因,实现了沙门氏菌dptC基因的高通量表达;表达载体中引入TEV酶切位点,使得很容易切除GST标签,为进一步研究DptC的酶学功能奠定了基础;沙门氏菌DptC发酵体系中添加50μmol/L Fe2+可以提高DptC产量,纯化的DptC显示浅棕色,推测与变铅青链霉菌(Streptomyces lividans)中的同源蛋白蛋白DndC一样,也是一种含4Fe-4S的铁硫蛋白。
[Objective] DNA phosphorothioate modification means substituting a non-bridging oxygen with a sulfur in DNA. The modification endows DNA with such chemical property that protects the hosting bacteria against peroxide. The modification is controlled by a dnd gene cluster. Salmonella entericaserovar Cerro 87 is one of the bacteria that harbor the DNA phosphorothioate modification. The modification is carried out by dptB,C,DandE. Ourstudy is designed to clone and express dptC,to optimize the expressing condition,and then to purify the DptC. [Methods]dptC DNA fragment was amplified by KOD PCR with the special primers and S. entericaserovar Cerro 87 genomic DNA template. A fusion expression vector pJTU3622 was constructed by inserting the dptC DNA fragment into pGEX-6P-1 inSmaI and XhoI sites.The positive clone was verified by antibiotics resistance gene screening and sequenced,and then transferred into host strain E. coli BL21( DE3) pLysS to producean engineering bacterium Anxh103. After optimizing the expression condition for dptC,we purified DptC from Anxh103 by kta FPLC with a GST-Trap column. [Results] A fusion expression vector pJTU3622 and an engineering bacterium Anxh103 were produced. The optimizing expressing condition for dptC is as follows: induced at 18℃ for 8- 18 h; 0. 6 mmol / L IPTG,LB with 50 μmol / L Fe2 +. [Conclusion] The anchor redeemed for high throughput expression of dptC. The TEV site in pJTU3622 made the process of purifying DptC easier and simpler. This helps lay the ground work for future study on the function of DptC. Also,the light brown color of DptC and the medium with 50 μmol / L Fe2 +showed us DptChas the same character with DndC which belongs to an iron-sufur protein with 4Fe- 4S.
[Objective] DNA phosphorothioate modification means substituting a non-bridging oxygen with a sulfur in DNA. The modification endows DNA with such chemical property that protects the hosting bacteria against peroxide. The modification is controlled by a dnd gene cluster. Salmonella entericaserovar Cerro 87 is one of the bacteria that harbor the DNA phosphorothioate modification. The modification is carried out by dptB,C,DandE. Ourstudy is designed to clone and express dptC,to optimize the expressing condition,and then to purify the DptC. [Methods]dptC DNA fragment was amplified by KOD PCR with the special primers and S. entericaserovar Cerro 87 genomic DNA template. A fusion expression vector pJTU3622 was constructed by inserting the dptC DNA fragment into pGEX-6P-1 inSmaI and XhoI sites.The positive clone was verified by antibiotics resistance gene screening and sequenced,and then transferred into host strain E. coli BL21( DE3) pLysS to producean engineering bacterium Anxh103. After optimizing the expression condition for dptC,we purified DptC from Anxh103 by kta FPLC with a GST-Trap column. [Results] A fusion expression vector pJTU3622 and an engineering bacterium Anxh103 were produced. The optimizing expressing condition for dptC is as follows: induced at 18℃ for 8- 18 h; 0. 6 mmol / L IPTG,LB with 50 μmol / L Fe2 +. [Conclusion] The anchor redeemed for high throughput expression of dptC. The TEV site in pJTU3622 made the process of purifying DptC easier and simpler. This helps lay the ground work for future study on the function of DptC. Also,the light brown color of DptC and the medium with 50 μmol / L Fe2 +showed us DptChas the same character with DndC which belongs to an iron-sufur protein with 4Fe- 4S.
引文
[1]Mckee T,Mckee JR.Biochemistry:An introduction.2nd eds.New York:McGraw-Hill Companies,Inc.2000.
[2]Brown TA.Genomes.2ndeds.Oxford:Wiley-Liss,2002.
[3]郑集,陈钧辉.普通生物化学.第四版.北京:高等教育出版社,2007.
[4]Zhou X,He X,Liang J,Li A,Xu T,Kieser T,Helmann JD,Deng Z.A novel DNA modification by sulphur.Molecular Microbiology,2005,57(5):1428-1438.
[5]Ou H,He X,Shao Y,Tai C,Rajakumar K,Deng Z.dnd DB:A Database Focused on Phosphorothioation of the DNA Backbone.PLoS One,2009,4(4):e5132.
[6]Wang L,Chen S,Vergin KL,Giovannoni SJ,Chan SW,DeMott MS,Taghizadeh K,Cordero OX,Cutler M,Timberlake S,Alm EJ,Polz MF,Pinhassi J,Deng Z,Dedon PC.DNA phosphorothioation is widespread and quantized in bacterial genomes.Proceedings of the National Academy of Sciences,2011,108(7):2963-2968.
[7]Zhou,X,Deng Z,Firmin JL,Hopwood DA,Kieser T.Site-specific degradation of Streptomyces lividans DNA during electrophoresis in buffers contaminated with ferrous iron.Nucleic Acids Research,1988,16(10):4341-4352.
[8]Wang L,Chen S,Xu T,Taghizadeh K,Wishnok JS,Zhou X,You D,Deng Z,Dedon PC.Phosphorothioation of DNA in bacteria by dnd genes.Nature Chemical Biology,2007,3(11):709-710.
[9]Xu T,Yao F,Zhou X,Deng Z,You D.A novel hostspecific restriction system associated with DNA backbone S-modification in Salmonella.Nucleic Acids Research,2010,38(20):7133-7141.
[10]Liu G,Ou H,Wang T,Li L,Tan H,Zhou X,Rajakumar K,Deng Z,He X.Cleavage of phosphorothioated DNA and methylated DNA by the type IV restriction endonuclease ScoMcrA.PLoS Genetics,2010,6(12):e1001253.
[11]Xie X,Liang J,Pu T,Xu F,Yao F,Yang Y,Zhao Yi,You D,Zhou X,Deng Z,Wang Z.Phosphorothioate DNA as an antioxidant in bacteria.Nucleic Acids Research,2012:1-10.
[12]He X,Ou H,Yu Q,Zhou X,Wu J,Liang J,Zhang W,Rajakumar K,Deng Z.Analysis of a genomic island housing genes for DNA S-modification system in Streptomyces lividans 66 and its counterparts in other distantly related bacteria.Molecular Microbiology,2007,65:1034-1048.
[13]Zhou X,He X,Li A,Lei F,s Kieser T,Deng Z.Streptomyces coelicolor A3(2)lacks a genomic island present in the chromosome of Streptomyces lividans 66.Applied and Environmental Microbiology,2004,70(12):7110-7118.
[14]You D,Wang L,Yao F,Zhou X,Deng Z.A novel DNA modification by sulfur:DndA is a NifS-like cysteine desulfurase capable of assembling DndC as an iron-sulfur cluster protein in Streptomyces lividans.Biochemistry,2007,46:6126-6133.
[15]An X,Xiong W,Yang Y,Li F,Zhou X,Wang Z,Deng Z,Liang J.A novel target of IscS in Escherichia coli:participating in DNA phosphorothioation.PLoS One,7(12):e51265.doi:10.1371/journal.pone.0051265.
[16]汪家政,范明.蛋白质技术手册.北京:科学出版社,2000.
[17]Xu T,Liang J,Chen S,Wang L,He X,You D,Wang Z,Li A,Xu Z,Zhou X,Deng Z.DNA phosphorothioation in Streptomyces lividans:mutational analysis of the dnd locus.BMC Microbiology,2009,9:41.
[18]An X,Zhou X,Wang Z,Deng Z,Liang J.Mutagenesis of cysteine residues in dptC from Salmonella enteric serovar Cerro 87 and its effects on DNA phosphorothioate modification.Acta Microbiological Sinica,2013,53(2):204-209.(in Chinese)安贤惠,周秀芬,汪志军,邓子新,梁晶丹.Salmonella enterica硫修饰蛋白DptC半胱氨酸定点突变对Dnd表型的影响.微生物学报,2013,53(2):204-209.
[2]Brown TA.Genomes.2ndeds.Oxford:Wiley-Liss,2002.
[3]郑集,陈钧辉.普通生物化学.第四版.北京:高等教育出版社,2007.
[4]Zhou X,He X,Liang J,Li A,Xu T,Kieser T,Helmann JD,Deng Z.A novel DNA modification by sulphur.Molecular Microbiology,2005,57(5):1428-1438.
[5]Ou H,He X,Shao Y,Tai C,Rajakumar K,Deng Z.dnd DB:A Database Focused on Phosphorothioation of the DNA Backbone.PLoS One,2009,4(4):e5132.
[6]Wang L,Chen S,Vergin KL,Giovannoni SJ,Chan SW,DeMott MS,Taghizadeh K,Cordero OX,Cutler M,Timberlake S,Alm EJ,Polz MF,Pinhassi J,Deng Z,Dedon PC.DNA phosphorothioation is widespread and quantized in bacterial genomes.Proceedings of the National Academy of Sciences,2011,108(7):2963-2968.
[7]Zhou,X,Deng Z,Firmin JL,Hopwood DA,Kieser T.Site-specific degradation of Streptomyces lividans DNA during electrophoresis in buffers contaminated with ferrous iron.Nucleic Acids Research,1988,16(10):4341-4352.
[8]Wang L,Chen S,Xu T,Taghizadeh K,Wishnok JS,Zhou X,You D,Deng Z,Dedon PC.Phosphorothioation of DNA in bacteria by dnd genes.Nature Chemical Biology,2007,3(11):709-710.
[9]Xu T,Yao F,Zhou X,Deng Z,You D.A novel hostspecific restriction system associated with DNA backbone S-modification in Salmonella.Nucleic Acids Research,2010,38(20):7133-7141.
[10]Liu G,Ou H,Wang T,Li L,Tan H,Zhou X,Rajakumar K,Deng Z,He X.Cleavage of phosphorothioated DNA and methylated DNA by the type IV restriction endonuclease ScoMcrA.PLoS Genetics,2010,6(12):e1001253.
[11]Xie X,Liang J,Pu T,Xu F,Yao F,Yang Y,Zhao Yi,You D,Zhou X,Deng Z,Wang Z.Phosphorothioate DNA as an antioxidant in bacteria.Nucleic Acids Research,2012:1-10.
[12]He X,Ou H,Yu Q,Zhou X,Wu J,Liang J,Zhang W,Rajakumar K,Deng Z.Analysis of a genomic island housing genes for DNA S-modification system in Streptomyces lividans 66 and its counterparts in other distantly related bacteria.Molecular Microbiology,2007,65:1034-1048.
[13]Zhou X,He X,Li A,Lei F,s Kieser T,Deng Z.Streptomyces coelicolor A3(2)lacks a genomic island present in the chromosome of Streptomyces lividans 66.Applied and Environmental Microbiology,2004,70(12):7110-7118.
[14]You D,Wang L,Yao F,Zhou X,Deng Z.A novel DNA modification by sulfur:DndA is a NifS-like cysteine desulfurase capable of assembling DndC as an iron-sulfur cluster protein in Streptomyces lividans.Biochemistry,2007,46:6126-6133.
[15]An X,Xiong W,Yang Y,Li F,Zhou X,Wang Z,Deng Z,Liang J.A novel target of IscS in Escherichia coli:participating in DNA phosphorothioation.PLoS One,7(12):e51265.doi:10.1371/journal.pone.0051265.
[16]汪家政,范明.蛋白质技术手册.北京:科学出版社,2000.
[17]Xu T,Liang J,Chen S,Wang L,He X,You D,Wang Z,Li A,Xu Z,Zhou X,Deng Z.DNA phosphorothioation in Streptomyces lividans:mutational analysis of the dnd locus.BMC Microbiology,2009,9:41.
[18]An X,Zhou X,Wang Z,Deng Z,Liang J.Mutagenesis of cysteine residues in dptC from Salmonella enteric serovar Cerro 87 and its effects on DNA phosphorothioate modification.Acta Microbiological Sinica,2013,53(2):204-209.(in Chinese)安贤惠,周秀芬,汪志军,邓子新,梁晶丹.Salmonella enterica硫修饰蛋白DptC半胱氨酸定点突变对Dnd表型的影响.微生物学报,2013,53(2):204-209.