黄鳝肠道假单胞杆菌铁蛋白基因的克隆及融合表达
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摘要
细菌铁蛋白(bacterioferritin,Bfr)在细菌铁代谢过程中具重要作用。为分析黄鳝肠道假单胞杆菌铁蛋白基因Bfr的功能,以该菌基因组DNA为模板,PCR扩增Bfr基因后将其克隆到pGEX-4T-1表达载体中。序列测定后转化大肠埃希菌BL21(DE3),经IPTG诱导、GST-resin纯化柱分离纯化获得Bfr蛋白。用SDS-PAGE电泳及Western blot检测融合蛋白;用比色法检测融合蛋白的铁离子螯合能力,用液体培养法分析重组蛋白对假单胞杆菌生长的影响。实验结果表明:成功获得黄鳝肠道假单胞杆菌Bfr基因,并实现了Bfr基因的融合表达;带有融合标签的GST-Bfr蛋白与Fe~(3+)和Fe~(2+)均不能结合,而Bfr蛋白则可以结合Fe~(3+)但不能结合Fe~(2+);在外源添加Fe~(3+)情况下,重组Bfr蛋白可促进假单胞杆菌的营养生长。该研究为进一步深入了解假单胞杆菌铁蛋白基因的功能提供了参考资料。
Bacterioferritin( Bfr) is a member of the ferritin family and plays an important role in intracellular iron metabolism. In order to probe the function of the Bfr gene,the g DNA of Pseudomonas spp. was isolated and used as templates to amplify the Bfr gene by PCR. Subsequently,the sequenced fragments were subcloned into pGEX-4 T-1 vector. The recombinant vector was transformed into E. coli BL21( DE3) and induced by IPTG. The GST-Bfr protein and Bfr protein were isolated and purified by GST-resin purification column,and then identified by Western blot. Finally,iron chelation examination was investigated. The results showed that the prokaryotic expression vector pGEX-4 T-1-Bfr was successfully constructed. SDS-PAGE and Western blot displayed that fusion protein GST-Bfr and Bfr were both purified. Iron chelation assay suggested that GST-Bfr protein could not bind Fe~(3+)and Fe~(2+),while Bfr could bind Fe~(3+)but could not bind Fe~(2+). Liquid culture experiments further showed that recombinant Bfr could promote the vegetative growth of Pseudomonas spp. to a certain extent. These results provide a theoretical basis for further study of gene function of bacterioferritin.
Bacterioferritin( Bfr) is a member of the ferritin family and plays an important role in intracellular iron metabolism. In order to probe the function of the Bfr gene,the g DNA of Pseudomonas spp. was isolated and used as templates to amplify the Bfr gene by PCR. Subsequently,the sequenced fragments were subcloned into pGEX-4 T-1 vector. The recombinant vector was transformed into E. coli BL21( DE3) and induced by IPTG. The GST-Bfr protein and Bfr protein were isolated and purified by GST-resin purification column,and then identified by Western blot. Finally,iron chelation examination was investigated. The results showed that the prokaryotic expression vector pGEX-4 T-1-Bfr was successfully constructed. SDS-PAGE and Western blot displayed that fusion protein GST-Bfr and Bfr were both purified. Iron chelation assay suggested that GST-Bfr protein could not bind Fe~(3+)and Fe~(2+),while Bfr could bind Fe~(3+)but could not bind Fe~(2+). Liquid culture experiments further showed that recombinant Bfr could promote the vegetative growth of Pseudomonas spp. to a certain extent. These results provide a theoretical basis for further study of gene function of bacterioferritin.
引文
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[11]RUVINSKY A M,VAKSER I A,RIVERA M. Local packing modulates diversity of iron pathways and cooperative behavior in eukaryotic and prokaryotic ferritins[J]. Journal of Chemical Physics,2014,140(11):115104.
[12]KRYACHKO Y,SEMLER D,VOGRINETZ J,et al. Enrichment and identification of biosurfactant-producing oil field microbiota utilizing electron acceptors other than oxygen and nitrate[J]. Journal of Biotechnology,2016,231:9-15.
[13]KEREN N,AURORA R,PAKRASI H B. Critical roles of bacterioferritins in iron storage and proliferation of cyanobacteria[J]. Plant Physiology,2004,135(3):1666-1673.
[14]WALTER P B,KNUTSON M D,PALER-MARTINEZ A,et al. Iron deficiency and iron excess damage mitochondria and mitochondrial DNA in rats[J]. Proceedings of the National Academy of Sciences of the United States of America,2002,99(4):2264-2269.
[15]LISIECKI P. Transferrin and lactoferrin-human iron sources for enterococci[J]. Polish Journal of Microbiology,2017,66(4):419-425.
[16]NAM K H,XU Y,PIAO S,et al. Crystal structure of bacterioferritin from Rhodobacter sphaeroides[J]. Biochemical&Biophysical Research Communications,2010,391(1):990-994.
[17]ROSA L,CUTONE A,LEPANTO M S,et al. Lactoferrin:a natural glycoprotein involved in iron and inflammatory homeostasis[J]. International Journal of Molecular Sciences,2017,18(9):1985.
[18]WAHLGREN W Y,OMRAN H,STETTEN D V,et al. Structural characterization of bacterioferritin from Blastochloris viridis[J]. Plo S One,2012,7(10):e46992.
[2]ESHELMAN K,Yao H,PUNCHI HEWAGE A N D et al. Inhibiting the BfrB:Bfd interaction in Pseudomonas aeruginosacauses irreversible iron accumulation in bacterioferritin and iron deficiency in the bacterial cytosol[J]. Metallomics,2017,9(6):646-659.
[3]RIVERA M. Bacterioferritin:structure,dynamics,and protein-protein interactions at play in iron storage and mobilization[J]. Accounts of Chemical Research,2017,50(2):331-340.
[4]ANDREWS S C. The Ferritin-like superfamily:evolution of the biological iron storeman from a ruberythrin-like ancestor[J]. Biochimica et Biophysica Acta,2010,1800(8):691-705.
[5]THEIL E C,TOSHA T,BEHERA R K. Solving biology’s iron chemistry problem with ferritin protein nanocages[J]. Accounts of Chemical Research,2016,49(5):784-791.
[6]BRADLEY J M,LE BRUN N E,MOORE G R. Ferritins:furnishing proteins with iron[J]. Journal of Biological Inorganic Chemistry,2016,21(1):13-28.
[7]YAO H,JEPKORIR G,LOVELL S,et al. Two distinct ferritin-like molecules in Pseudomonas aeruginosa:the product of the bfr A gene is a bacterial ferritin(Ftn A)and not a bacterioferritin(Bfr)[J]. Biochemistry,2011,50(23):5236-5248.
[8]CROW A,LAWSON T L,LEWIN A,et al. Structural basis for iron mineralization by bacterioferritin[J]. Journal of American Chemical Society,2009,131(19):6808-6813.
[9]WEERATUNGA S,GEE C E,LOVELL S,et al. Binding of Pseudomonas aeruginosa apobacterioferritin-associated ferredoxin to bacterioferritin B promotes heme mediation of electron delivery and mobilization of core mineral iron[J]. Biochemistry,2009,48(31):7420-7431.
[10]BRADLEY J M,SVISTUNENKO D A,MOORE G R,et al.Tyr25,Tyr58 and Trp133 of Escherichia coli bacterioferritin transfer electrons between iron in the central cavity and the ferroxidase centre[J]. Metallomics,2017,9(10):1421-1428.
[11]RUVINSKY A M,VAKSER I A,RIVERA M. Local packing modulates diversity of iron pathways and cooperative behavior in eukaryotic and prokaryotic ferritins[J]. Journal of Chemical Physics,2014,140(11):115104.
[12]KRYACHKO Y,SEMLER D,VOGRINETZ J,et al. Enrichment and identification of biosurfactant-producing oil field microbiota utilizing electron acceptors other than oxygen and nitrate[J]. Journal of Biotechnology,2016,231:9-15.
[13]KEREN N,AURORA R,PAKRASI H B. Critical roles of bacterioferritins in iron storage and proliferation of cyanobacteria[J]. Plant Physiology,2004,135(3):1666-1673.
[14]WALTER P B,KNUTSON M D,PALER-MARTINEZ A,et al. Iron deficiency and iron excess damage mitochondria and mitochondrial DNA in rats[J]. Proceedings of the National Academy of Sciences of the United States of America,2002,99(4):2264-2269.
[15]LISIECKI P. Transferrin and lactoferrin-human iron sources for enterococci[J]. Polish Journal of Microbiology,2017,66(4):419-425.
[16]NAM K H,XU Y,PIAO S,et al. Crystal structure of bacterioferritin from Rhodobacter sphaeroides[J]. Biochemical&Biophysical Research Communications,2010,391(1):990-994.
[17]ROSA L,CUTONE A,LEPANTO M S,et al. Lactoferrin:a natural glycoprotein involved in iron and inflammatory homeostasis[J]. International Journal of Molecular Sciences,2017,18(9):1985.
[18]WAHLGREN W Y,OMRAN H,STETTEN D V,et al. Structural characterization of bacterioferritin from Blastochloris viridis[J]. Plo S One,2012,7(10):e46992.