嗜根考克氏菌翻译延伸因子P基因的克隆、表达及其条件优化
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摘要
为获得大量可溶性重组蛋白EF-P用以构建以EF-P蛋白为靶标的新型、高效抗细菌抗生素筛选模型,研究嗜根考克氏菌DC2201 efp基因的体外克隆、表达及表达条件的优化.首先对efp基因进行生物信息学分析,随后经PCR特异性扩增获得efp基因并将其插入表达质粒pET-29a(+)中,重组质粒(pET-29a(+)-efp)转化至表达宿主菌株E.coli BL21(DE3)进行诱导表达,通过优化诱导表达条件(起始菌体浓度、温度、IPTG终浓度、时间)获得大量可溶性目的蛋白,最后对表达产物进行镍离子亲和层析柱纯化及SDS-PAGE、MALDI-TOF-TOF鉴定.结果获得相对分子质量大小约为25 kDa的蛋白条带,与预测的目的重组蛋白相对分子量大小相符.选择O_(D600 nm)
In order to obtain a large number of soluble recombinant protein EF-P to construct a novel and highly effective antibiotic screening model targeting EF-P protein,the efp gene from Kocuria rhizophila DC2201 is cloned and expressed and the expression conditions is optimized.At first,efp is analyzed by bioinformatics method.Subsequently,the gene is amplified by PCR and inserted into the expression vector pET-29 a(+).The recombinant plasmid(pET-29 a(+)-efp) is transformed into the expression host strain E.coli BL21(DE3) to induce its expression.It can obtain many target proteins by optimizing the expression conditions(initial bacterial density,induction temperature,IPTG final concentration,induction time).Finally,the expression products is purified by nickel ion affinity chromatography and identified by SDS-PAGE and MALDI-TOF-TOF.A protein band with approximately 25 kDa is showed,which is consistent with the predicted molecular weight of recombinant protein.Under O_(D600 nm)
In order to obtain a large number of soluble recombinant protein EF-P to construct a novel and highly effective antibiotic screening model targeting EF-P protein,the efp gene from Kocuria rhizophila DC2201 is cloned and expressed and the expression conditions is optimized.At first,efp is analyzed by bioinformatics method.Subsequently,the gene is amplified by PCR and inserted into the expression vector pET-29 a(+).The recombinant plasmid(pET-29 a(+)-efp) is transformed into the expression host strain E.coli BL21(DE3) to induce its expression.It can obtain many target proteins by optimizing the expression conditions(initial bacterial density,induction temperature,IPTG final concentration,induction time).Finally,the expression products is purified by nickel ion affinity chromatography and identified by SDS-PAGE and MALDI-TOF-TOF.A protein band with approximately 25 kDa is showed,which is consistent with the predicted molecular weight of recombinant protein.Under O_(D600 nm)
引文
[1] Zinner S H.Antibiotic use:present and future [J].New Microbiology,2007,30(3):321-325.
[2] Dan I A.Persistence of antibiotic resistant bacteria [J].Current Opinion in Microbiology,2003,6(5):452-456.
[3] Doerfel L K,Rodnina M V.Elongation factor P:function and effects on bacterial fitness [J].Biopolymers,2013,99(11):837-845.
[4] Doerfel L K,Wohlgemuth I,Kothe C,et al.EF-P is essential for rapid synthesis of proteins containing consecutive proline residues [J].Science,2013,339(6115):85-88.
[5] Ude S,Lassak J,Starosta A L,et al.Translation elongation factor EF-P alleviates ribosome stalling at polyproline stretches [J].Science,2013,339(6115):82-85.
[6] Lassak J,Wilson D N,Jung K.Stall no more at polyproline stretches with the translation elongation factors EF-P and IF-5A [J].Molecular Microbiology,2016,99(2):219-235.
[7] Zou S B,Hersch S J,Roy H,et al.Loss of elongation factor P disrupts bacterial outer membrane integrity [J].Journal of Bacteriology,2012,194(2):413-425.
[8] Kaniga K,Compton M S,Curtiss R,et al.Molecular and functional characterization of Salmonella enterica serovar typhimurium poxA gene:effect on attenuation of virulence and protection [J].Infection and Immunity,1998,66(12):5599-5606.
[9] Charles T C,Nester E W.A chromosomally encoded two-component sensory transduction system is required for virulence of Agrobacterium tumefaciens [J].Journal of Bacteriology,1993,175(20):6614-6625.
[10] Peng Wentao,Banta L M,Charles T C,et al.The chvH locus of Agrobacterium Encodes a homologue of an elongation factor involved in protein synthesis [J].Journal of Bacteriology,2001,183(1):36-45.
[11] Florencia I,Ugalde J E,Nora I D I.Brucella abortus efp gene is required for an efficient internalization in HeLa cells [J].Microbial Pathogenesis,2012,52(1):31-40.
[12] Bearson S M,Bearson B L,Brunelle B W,et al.A mutation in the poxA gene of Salmonella enterica serovar typhimurium alters protein production,elevates susceptibility to environmental challenges,and decreases swine colonization [J].Foodborne Pathogens and Disease,2011,8(8):725-732.
[13] Dyk T K V,Smulski D R,Chang Yingying.Pleiotropic effects of poxA regulatory mutations of Escherichia coli and Salmonella typhimurium,mutations conferring sulfometuron methyl and alpha-ketobutyrate hypersensitivity [J].Journal of Bacteriology,1987,169(10):4540-4546.
[14] Swaney S,Mccroskey M,Shinabarger D,et al.Characterization of a high-throughput screening assay for inhibitors of elongation factor p and ribosomal peptidyl transferase activity [J].Journal of Biomolecular Screening,2006,11(7):736.
[15] Takarada H,Sekine M,Kosugi H,et al.Complete genome sequence of the soil actinomycete Kocuria rhizophila [J].Journal of Bacteriology,2008,190(12):4139-4146.
[16] 冯思玲.系统发育树构建方法研究 [J].信息技术,2009(6):38-40.
[17] Bullwinkle T J,Zou S B,Rajkovic A,et al.(R)-β-lysine-modified elongation factor P functions in translation elongation [J].Journal of Biological Chemistry,2013,288(6):4416.
[18] Rajkovic A,Erickson S,Witzky A,et al.Cyclic rhamnosylated elongation factor P establishes antibiotic resistance in Pseudomonas aeruginosa [J].Mbio,2015,6(3):155-170.
[19] Payne R J,Wang Siyao,Corcilius L,et al.Synthesis of rhamnosylated arginine glycopeptides and determination of the glycosidic linkage in bacterial elongation factor P [J].Chemical Science,2017,8(3):2296.
[20] Lassak J,Keilhauer E C,Fürst M,et al.Corrigendum:arginine-rhamnosylation as new strategy to activate translation elongation factor P [J].Nature Chemical Biology,2015,11(4):266-270.
[21] Balibar C J,Iwanowicz D,Dean C R.Elongation factor P is dispensable in Escherichia coli and Pseudomonas aeruginosa [J].Current Microbiology,2013,67(3):293-299.
[22] Choi E,Choi S,Nam D,et al.Elongation factor P restricts Salmonella′s growth by controlling translation of a Mg2+ transporter gene during infection [J].Sci Rep,2017,7:42098.
[23] Navarre W W,Zou S,Roy H,et al.PoxA, YjeK and elongation factor P coordinately modulate virulence and drug resistance in Salmonella enterica [J].Molecular Cell,2010,39(2):209-221.
[24] Aoki H,Xu J,Emili A,et al.Interactions of elongation factor EF-P with the Escherichia coli ribosome [J].Febs Journal,2008,275(4):671-681.
[25] Mohapatra S,Choi H,Ge Xueliang,et al.Spatial distribution and ribosome-binding dynamics of EF-P in live Escherichia coli [J].Mbio,2017,8(3):e00300-17.
[26] Ude S,Lassak J,Starosta A L,et al.Translation elongation factor EF-P alleviates ribosome stalling at polyproline stretches [J].Science,2013,339(6115):82-85.
[27] Peil L,Starosta A L,Lassak J,et al.Distinct XPPX sequence motifs induce ribosome stalling,which is rescued by the translation elongation factor EF-P [J].PNAS,2013,110(38):15265-15270.
[28] Rajkovic A,Ibba M.Elongation factor P and the control of translation elongation [J].Annual Review of Microbiology,2017,71(1):117-131.
[2] Dan I A.Persistence of antibiotic resistant bacteria [J].Current Opinion in Microbiology,2003,6(5):452-456.
[3] Doerfel L K,Rodnina M V.Elongation factor P:function and effects on bacterial fitness [J].Biopolymers,2013,99(11):837-845.
[4] Doerfel L K,Wohlgemuth I,Kothe C,et al.EF-P is essential for rapid synthesis of proteins containing consecutive proline residues [J].Science,2013,339(6115):85-88.
[5] Ude S,Lassak J,Starosta A L,et al.Translation elongation factor EF-P alleviates ribosome stalling at polyproline stretches [J].Science,2013,339(6115):82-85.
[6] Lassak J,Wilson D N,Jung K.Stall no more at polyproline stretches with the translation elongation factors EF-P and IF-5A [J].Molecular Microbiology,2016,99(2):219-235.
[7] Zou S B,Hersch S J,Roy H,et al.Loss of elongation factor P disrupts bacterial outer membrane integrity [J].Journal of Bacteriology,2012,194(2):413-425.
[8] Kaniga K,Compton M S,Curtiss R,et al.Molecular and functional characterization of Salmonella enterica serovar typhimurium poxA gene:effect on attenuation of virulence and protection [J].Infection and Immunity,1998,66(12):5599-5606.
[9] Charles T C,Nester E W.A chromosomally encoded two-component sensory transduction system is required for virulence of Agrobacterium tumefaciens [J].Journal of Bacteriology,1993,175(20):6614-6625.
[10] Peng Wentao,Banta L M,Charles T C,et al.The chvH locus of Agrobacterium Encodes a homologue of an elongation factor involved in protein synthesis [J].Journal of Bacteriology,2001,183(1):36-45.
[11] Florencia I,Ugalde J E,Nora I D I.Brucella abortus efp gene is required for an efficient internalization in HeLa cells [J].Microbial Pathogenesis,2012,52(1):31-40.
[12] Bearson S M,Bearson B L,Brunelle B W,et al.A mutation in the poxA gene of Salmonella enterica serovar typhimurium alters protein production,elevates susceptibility to environmental challenges,and decreases swine colonization [J].Foodborne Pathogens and Disease,2011,8(8):725-732.
[13] Dyk T K V,Smulski D R,Chang Yingying.Pleiotropic effects of poxA regulatory mutations of Escherichia coli and Salmonella typhimurium,mutations conferring sulfometuron methyl and alpha-ketobutyrate hypersensitivity [J].Journal of Bacteriology,1987,169(10):4540-4546.
[14] Swaney S,Mccroskey M,Shinabarger D,et al.Characterization of a high-throughput screening assay for inhibitors of elongation factor p and ribosomal peptidyl transferase activity [J].Journal of Biomolecular Screening,2006,11(7):736.
[15] Takarada H,Sekine M,Kosugi H,et al.Complete genome sequence of the soil actinomycete Kocuria rhizophila [J].Journal of Bacteriology,2008,190(12):4139-4146.
[16] 冯思玲.系统发育树构建方法研究 [J].信息技术,2009(6):38-40.
[17] Bullwinkle T J,Zou S B,Rajkovic A,et al.(R)-β-lysine-modified elongation factor P functions in translation elongation [J].Journal of Biological Chemistry,2013,288(6):4416.
[18] Rajkovic A,Erickson S,Witzky A,et al.Cyclic rhamnosylated elongation factor P establishes antibiotic resistance in Pseudomonas aeruginosa [J].Mbio,2015,6(3):155-170.
[19] Payne R J,Wang Siyao,Corcilius L,et al.Synthesis of rhamnosylated arginine glycopeptides and determination of the glycosidic linkage in bacterial elongation factor P [J].Chemical Science,2017,8(3):2296.
[20] Lassak J,Keilhauer E C,Fürst M,et al.Corrigendum:arginine-rhamnosylation as new strategy to activate translation elongation factor P [J].Nature Chemical Biology,2015,11(4):266-270.
[21] Balibar C J,Iwanowicz D,Dean C R.Elongation factor P is dispensable in Escherichia coli and Pseudomonas aeruginosa [J].Current Microbiology,2013,67(3):293-299.
[22] Choi E,Choi S,Nam D,et al.Elongation factor P restricts Salmonella′s growth by controlling translation of a Mg2+ transporter gene during infection [J].Sci Rep,2017,7:42098.
[23] Navarre W W,Zou S,Roy H,et al.PoxA, YjeK and elongation factor P coordinately modulate virulence and drug resistance in Salmonella enterica [J].Molecular Cell,2010,39(2):209-221.
[24] Aoki H,Xu J,Emili A,et al.Interactions of elongation factor EF-P with the Escherichia coli ribosome [J].Febs Journal,2008,275(4):671-681.
[25] Mohapatra S,Choi H,Ge Xueliang,et al.Spatial distribution and ribosome-binding dynamics of EF-P in live Escherichia coli [J].Mbio,2017,8(3):e00300-17.
[26] Ude S,Lassak J,Starosta A L,et al.Translation elongation factor EF-P alleviates ribosome stalling at polyproline stretches [J].Science,2013,339(6115):82-85.
[27] Peil L,Starosta A L,Lassak J,et al.Distinct XPPX sequence motifs induce ribosome stalling,which is rescued by the translation elongation factor EF-P [J].PNAS,2013,110(38):15265-15270.
[28] Rajkovic A,Ibba M.Elongation factor P and the control of translation elongation [J].Annual Review of Microbiology,2017,71(1):117-131.