Synthesis and characteristic of a novel green fluorescent protein eYGFPuv
|
摘要
Recently, a novel green fluorescent protein eYGFPuv has been identified in the marine organism Chiridius poppei which displays high fluorescence intensity and can be visible by eyes in dark. Although strong green fluorescence was achieved in transgenic petunia, 3 expression cassettes(about 8 kb) complicate its application. In this study, to confirm whether 1 expression cassette could be used as a transgenic marker in prokaryotes and eukaryotes, eYGFPuv was cloned into prokaryotic expression vector pET28α-eYGFPuv-His and plant binary expression vector 35 S::eYGFPuv. Compared to EGFP, eYGFPuv protein exhibited stronger dazzling green fluorescence in E. coli under excited light at 365 nm and maintains steadily over a long period of time without degradation. When transiently expressed in tobacco leaves, eYGFPuv protein displayed strong green fluorescence. Moreover, the fluorescence of eYGFPuv protein also could be directly observed in living plant, and thus can be used easily as a marker to screen transformed lines in transgenic research. Overall, compared to previous studies on eYGFPuv tandem repeats, our data confirmed that single eYGFPuv sequence still possesses high fluorescence intensity and quenching resistance. Furthermore, because of small size of expression cassette, it is suitable for efficient transformation in both prokaryotic and eukaryotic organisms.
Recently, a novel green fluorescent protein eYGFPuv has been identified in the marine organism Chiridius poppei which displays high fluorescence intensity and can be visible by eyes in dark. Although strong green fluorescence was achieved in transgenic petunia, 3 expression cassettes(about 8 kb) complicate its application. In this study, to confirm whether 1 expression cassette could be used as a transgenic marker in prokaryotes and eukaryotes, eYGFPuv was cloned into prokaryotic expression vector pET28α-eYGFPuv-His and plant binary expression vector 35 S::eYGFPuv. Compared to EGFP, eYGFPuv protein exhibited stronger dazzling green fluorescence in E. coli under excited light at 365 nm and maintains steadily over a long period of time without degradation. When transiently expressed in tobacco leaves, eYGFPuv protein displayed strong green fluorescence. Moreover, the fluorescence of eYGFPuv protein also could be directly observed in living plant, and thus can be used easily as a marker to screen transformed lines in transgenic research. Overall, compared to previous studies on eYGFPuv tandem repeats, our data confirmed that single eYGFPuv sequence still possesses high fluorescence intensity and quenching resistance. Furthermore, because of small size of expression cassette, it is suitable for efficient transformation in both prokaryotic and eukaryotic organisms.
Recently, a novel green fluorescent protein eYGFPuv has been identified in the marine organism Chiridius poppei which displays high fluorescence intensity and can be visible by eyes in dark. Although strong green fluorescence was achieved in transgenic petunia, 3 expression cassettes(about 8 kb) complicate its application. In this study, to confirm whether 1 expression cassette could be used as a transgenic marker in prokaryotes and eukaryotes, eYGFPuv was cloned into prokaryotic expression vector pET28α-eYGFPuv-His and plant binary expression vector 35 S::eYGFPuv. Compared to EGFP, eYGFPuv protein exhibited stronger dazzling green fluorescence in E. coli under excited light at 365 nm and maintains steadily over a long period of time without degradation. When transiently expressed in tobacco leaves, eYGFPuv protein displayed strong green fluorescence. Moreover, the fluorescence of eYGFPuv protein also could be directly observed in living plant, and thus can be used easily as a marker to screen transformed lines in transgenic research. Overall, compared to previous studies on eYGFPuv tandem repeats, our data confirmed that single eYGFPuv sequence still possesses high fluorescence intensity and quenching resistance. Furthermore, because of small size of expression cassette, it is suitable for efficient transformation in both prokaryotic and eukaryotic organisms.
引文
Bajar B.T.,Wang E.S.,Zhang S.,Lin M.Z.,Chu J.2016.Aguide to fluorescent protein FRET pairs.Sensors.16:1488.doi:10.3390/s16091488.
Chin D.P.,Shiratori I.,Shimizu A.,Kato K.,Mii M.,Waga I.2018.Generation of brilliant green fluorescent petunia plants by using a new and potent fluorescent protein transgene.Scientific Reports.8:16556.doi:10.1038/s41598-018-34837-2.
Cody C.W.,Prasher D.C.,Westler W.M.,Prendergast F.G.,Ward W.W.1993.Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein.Biochemistry.32(5):1212-1218.doi:10.1021/bi00056a003.
Crameri A.,Whitehorn E.A.,Tate E.,Stemmer W.P.C.1996.Improved green fluorescent protein by molecular evolution using DNA shuffling.Nature Biotechnology.14(3):315.doi:10.1038/nbt0396-315.
Ding X.,Chen Q.S.,Bao C.M.,Ai A.H.,Zhou Y.,Li S.B.,Xie H.W.,Zhu Y.L.,Cai Y.H.,Peng X.J.2016.Expression of a mitochondrial gene orfH79 from CMS-Honglian rice inhibits Escherichia coli growth via deficient oxygen consumption.SpringerPlus.5:1125.doi:10.1186/s40064-016-2822-0.
Fradkov A.F.,Chen Y.,Ding L.,Barsova E.V.,Matz M.V.,Lukyanov S.A.2000.Novel fluorescent protein from Discosoma coral and its mutants possesses a unique far-red fluorescence.FEBS Letters.479(3):127-130.doi:10.1016/s0014-5793(00)01895-0.
Gong Z.,Wan H.,Tay T.L.,Wang H.,Chen M.,Yan T.2003.Development of transgenic fish for ornamental and bioreactor by strong expression of fluorescent proteins in the skeletal muscle.Biochemical and Biophysical Research Communications.308(1):58-63.doi:10.1016/s0006-291x(03)01282-8.
Iizuka T.,Sezutsu H.,Tatematsu K.,Kobayashi I.,Yonemura N.,Uchino K.,Nakajima K.,Kojima K.,Takabayashi C.,Machii H.,et al.2013.Colored fluorescent silk made by transgenic silkworms.Advanced Functional Materials.23(42):5232-5239.doi:10.1002/adfm.201300365.
Ito Y.,Suzuki M.,Husimi Y.1999.A novel mutant of green fluorescent protein with enhanced sensitivity for microanalysis at 488 nm excitation.Biochemical and Biophysical Research Communications.264(2):556-560.doi:10.1006/bbrc.1999.1541.
Knop M.,Barr F.,Riedel C.,Heckel T.,Reichel C.2002.Improved version of the red fluorescent protein(drFP583/DsRed/RFP).BioTechniques.33(3):592-602.doi:10.2144/02333rr02.
Laviv T.,Kim B.B.,Chu J.,Lam A.J.,Lin M.Z.,Yasuda R.2016.Simultaneous dual-color fluorescence lifetime imaging with novel red-shifted fluorescent proteins.Nature Methods.13(12):989-992.doi:10.1038/nmeth.4046.
Mann D.G.,Abercrombie L.L.,Rudis M.R.,Millwood R.J.,Dunlap J.R.,Stewart C.N.2012.Very bright orange fluorescent plants:endoplasmic reticulum targeting of orange fluorescent proteins as visual reporters in transgenic plants.BMC Biotechnology.12(1):17.doi:10.1186/1472-6750-12-17.
Masuda H.,Takenaka Y.,Yamaguchi A.,Nishikawa S.,Mizuno H.2006.A novel yellowish-green fluorescent protein from the marine copepod,Chiridius poppei,and its use as a reporter protein in HeLa cells.Gene.372:18-25.doi:10.1016/j.gene.2005.11.031.
Niwa H.,Inouye S.,Hirano T.,Matsuno T.,Kojima S.,Kubota M.,Ohashi M.,Tsuji F.I.1996.Chemical nature of the light emitter of the Aequorea green fluorescent protein.Proceedings of the National Academy of Sciences.93(24):13617-13622.doi:10.1073/pnas.93.24.13617.
Rakosy T.E.,Aurori C.M.,Dijkstra C.,Thieme R.,Aurori A.,Davey M.R.2007.The usefulness of the gfp reporter gene for monitoring Agrobacterium-mediated transformation of potato dihaploid and tetraploid genotypes.Plant Cell Reports.26(5):661-671.doi:10.1007/s00299-006-0273-8.
Sasaki K.,Kato K.,Mishima H.,Furuichi M.,Waga I.,Takane K.,Yamaguchi H.,Ohtsubo N.2014.Generation of fluorescent flowers exhibiting strong fluorescence by combination of fluorescent protein from marine plankton and recent genetic tools in Torenia fournieri Lind.Plant Biotechnology.31(4):309-318.doi:10.5511/plantbiotechnology.14.0907a.
Shagin D.A.,Barsova E.V.,Yanushevich Y.G.,Fradkov A.F.,Lukyanov K.A.,Labas Y.A.,Semenova T.N.,Ugalde J.A.,Meyers A.,Nunez J.M.2004.GFP-like proteins as ubiquitous metazoan superfamily:evolution of functional features and structural complexity.Molecular Biology and Evolution.21(5):841-850.doi:10.1093/molbev/msh079.
Shaner N.C.,Steinbach P.A.,Tsien R.Y.2005.A guide to choosing fluorescent proteins.Nature Methods.2(12):905-909.doi:10.1038/nmeth819.
Shen Y.,Chen Y.,Wu J.,Shaner N.C.,Campbell R.E.2017.Engineering of mCherry variants with long Stokes shift,red-shifted fluorescence,and low cytotoxicity.PloS One.12(2):e0171257.doi:10.1371/journal.pone.0171257.
Shimizu A.,Shiratori I.,Horii K.,Waga I.2017.Molecular evolution of versatile derivatives from a GFP-like protein in the marine copepod Chiridius poppei.PloS One.12(7):e0181186.doi:10.1371/journal.pone.0181186.
Shimomura O.,Johnson F.H.,Saiga Y.1962.Extraction,purification and properties of aequorin,a bioluminescent protein from the luminous hydromedusan,Aequorea.Journal of Cellular and Comparative Physiology.59(3):223-239.doi:10.1002/jcp.1030590302.
Siemering K.R.,Golbik R.,Sever R.,Haseloff J.1996.Mutations that suppress the thermosensitivity of green fluorescent protein.Current Biology.6(12):1653-1663.doi:10.1016/S0960-9822(02)70789-6.
S?rensen M.,Lippuner C.,Kaiser T.,Mi?litz A.,Aebischer T.,Bumann D.2003.Rapidly maturing red fluorescent protein variants with strongly enhanced brightness in bacteria.FEBS Letters.552(213):110-114.doi:10.1016/S0014-5793(03)00856-1.
Stepanenko O.V.,Verkhusha V.V.,Kuznetsova I.M.,Uversky V.N.,Turoverov K.2008.Fluorescent proteins as biomarkers and biosensors:throwing color lights on molecular and cellular processes.Current Protein&Peptide Science.9(4):338-369.doi:10.2174/138920308785132668.
Suto K.,Masuda H.,Takenaka Y.,Tsuji F.I.,Mizuno H.2009.Structural basis for red-shifted emission of a GFP-like protein from the marine copepod Chiridius poppei.Genes to Cells.14(6):727-737.doi:10.1111/j.1365-2443.2009.01305.x.
Topell S.,Hennecke J.,Glockshuber R.1999.Circularly permuted variants of the green fluorescent protein.FEBS Letters.457(2):283-289.doi:10.1016/s0014-5793(99)01044-3.
Topping J.F.1998.Tobacco transformation.In:Plant Virology Protocols:Springer.p.365-372.doi:10.1385/0-89603-385-6:365.
Tsien R.Y.2006.Breeding and building molecules to spy on cells and tumors.The Keio Journal of Medicine.55(4):127-140.doi:10.2302/kjm.55.127.
Yang P.C.Mahmood T.,2012.Western blot:Technique,theory,and trouble shooting.North American Journal of Medical Sciences.4(9):429.doi:10.4103/1947-2714.100998.
Chin D.P.,Shiratori I.,Shimizu A.,Kato K.,Mii M.,Waga I.2018.Generation of brilliant green fluorescent petunia plants by using a new and potent fluorescent protein transgene.Scientific Reports.8:16556.doi:10.1038/s41598-018-34837-2.
Cody C.W.,Prasher D.C.,Westler W.M.,Prendergast F.G.,Ward W.W.1993.Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein.Biochemistry.32(5):1212-1218.doi:10.1021/bi00056a003.
Crameri A.,Whitehorn E.A.,Tate E.,Stemmer W.P.C.1996.Improved green fluorescent protein by molecular evolution using DNA shuffling.Nature Biotechnology.14(3):315.doi:10.1038/nbt0396-315.
Ding X.,Chen Q.S.,Bao C.M.,Ai A.H.,Zhou Y.,Li S.B.,Xie H.W.,Zhu Y.L.,Cai Y.H.,Peng X.J.2016.Expression of a mitochondrial gene orfH79 from CMS-Honglian rice inhibits Escherichia coli growth via deficient oxygen consumption.SpringerPlus.5:1125.doi:10.1186/s40064-016-2822-0.
Fradkov A.F.,Chen Y.,Ding L.,Barsova E.V.,Matz M.V.,Lukyanov S.A.2000.Novel fluorescent protein from Discosoma coral and its mutants possesses a unique far-red fluorescence.FEBS Letters.479(3):127-130.doi:10.1016/s0014-5793(00)01895-0.
Gong Z.,Wan H.,Tay T.L.,Wang H.,Chen M.,Yan T.2003.Development of transgenic fish for ornamental and bioreactor by strong expression of fluorescent proteins in the skeletal muscle.Biochemical and Biophysical Research Communications.308(1):58-63.doi:10.1016/s0006-291x(03)01282-8.
Iizuka T.,Sezutsu H.,Tatematsu K.,Kobayashi I.,Yonemura N.,Uchino K.,Nakajima K.,Kojima K.,Takabayashi C.,Machii H.,et al.2013.Colored fluorescent silk made by transgenic silkworms.Advanced Functional Materials.23(42):5232-5239.doi:10.1002/adfm.201300365.
Ito Y.,Suzuki M.,Husimi Y.1999.A novel mutant of green fluorescent protein with enhanced sensitivity for microanalysis at 488 nm excitation.Biochemical and Biophysical Research Communications.264(2):556-560.doi:10.1006/bbrc.1999.1541.
Knop M.,Barr F.,Riedel C.,Heckel T.,Reichel C.2002.Improved version of the red fluorescent protein(drFP583/DsRed/RFP).BioTechniques.33(3):592-602.doi:10.2144/02333rr02.
Laviv T.,Kim B.B.,Chu J.,Lam A.J.,Lin M.Z.,Yasuda R.2016.Simultaneous dual-color fluorescence lifetime imaging with novel red-shifted fluorescent proteins.Nature Methods.13(12):989-992.doi:10.1038/nmeth.4046.
Mann D.G.,Abercrombie L.L.,Rudis M.R.,Millwood R.J.,Dunlap J.R.,Stewart C.N.2012.Very bright orange fluorescent plants:endoplasmic reticulum targeting of orange fluorescent proteins as visual reporters in transgenic plants.BMC Biotechnology.12(1):17.doi:10.1186/1472-6750-12-17.
Masuda H.,Takenaka Y.,Yamaguchi A.,Nishikawa S.,Mizuno H.2006.A novel yellowish-green fluorescent protein from the marine copepod,Chiridius poppei,and its use as a reporter protein in HeLa cells.Gene.372:18-25.doi:10.1016/j.gene.2005.11.031.
Niwa H.,Inouye S.,Hirano T.,Matsuno T.,Kojima S.,Kubota M.,Ohashi M.,Tsuji F.I.1996.Chemical nature of the light emitter of the Aequorea green fluorescent protein.Proceedings of the National Academy of Sciences.93(24):13617-13622.doi:10.1073/pnas.93.24.13617.
Rakosy T.E.,Aurori C.M.,Dijkstra C.,Thieme R.,Aurori A.,Davey M.R.2007.The usefulness of the gfp reporter gene for monitoring Agrobacterium-mediated transformation of potato dihaploid and tetraploid genotypes.Plant Cell Reports.26(5):661-671.doi:10.1007/s00299-006-0273-8.
Sasaki K.,Kato K.,Mishima H.,Furuichi M.,Waga I.,Takane K.,Yamaguchi H.,Ohtsubo N.2014.Generation of fluorescent flowers exhibiting strong fluorescence by combination of fluorescent protein from marine plankton and recent genetic tools in Torenia fournieri Lind.Plant Biotechnology.31(4):309-318.doi:10.5511/plantbiotechnology.14.0907a.
Shagin D.A.,Barsova E.V.,Yanushevich Y.G.,Fradkov A.F.,Lukyanov K.A.,Labas Y.A.,Semenova T.N.,Ugalde J.A.,Meyers A.,Nunez J.M.2004.GFP-like proteins as ubiquitous metazoan superfamily:evolution of functional features and structural complexity.Molecular Biology and Evolution.21(5):841-850.doi:10.1093/molbev/msh079.
Shaner N.C.,Steinbach P.A.,Tsien R.Y.2005.A guide to choosing fluorescent proteins.Nature Methods.2(12):905-909.doi:10.1038/nmeth819.
Shen Y.,Chen Y.,Wu J.,Shaner N.C.,Campbell R.E.2017.Engineering of mCherry variants with long Stokes shift,red-shifted fluorescence,and low cytotoxicity.PloS One.12(2):e0171257.doi:10.1371/journal.pone.0171257.
Shimizu A.,Shiratori I.,Horii K.,Waga I.2017.Molecular evolution of versatile derivatives from a GFP-like protein in the marine copepod Chiridius poppei.PloS One.12(7):e0181186.doi:10.1371/journal.pone.0181186.
Shimomura O.,Johnson F.H.,Saiga Y.1962.Extraction,purification and properties of aequorin,a bioluminescent protein from the luminous hydromedusan,Aequorea.Journal of Cellular and Comparative Physiology.59(3):223-239.doi:10.1002/jcp.1030590302.
Siemering K.R.,Golbik R.,Sever R.,Haseloff J.1996.Mutations that suppress the thermosensitivity of green fluorescent protein.Current Biology.6(12):1653-1663.doi:10.1016/S0960-9822(02)70789-6.
S?rensen M.,Lippuner C.,Kaiser T.,Mi?litz A.,Aebischer T.,Bumann D.2003.Rapidly maturing red fluorescent protein variants with strongly enhanced brightness in bacteria.FEBS Letters.552(213):110-114.doi:10.1016/S0014-5793(03)00856-1.
Stepanenko O.V.,Verkhusha V.V.,Kuznetsova I.M.,Uversky V.N.,Turoverov K.2008.Fluorescent proteins as biomarkers and biosensors:throwing color lights on molecular and cellular processes.Current Protein&Peptide Science.9(4):338-369.doi:10.2174/138920308785132668.
Suto K.,Masuda H.,Takenaka Y.,Tsuji F.I.,Mizuno H.2009.Structural basis for red-shifted emission of a GFP-like protein from the marine copepod Chiridius poppei.Genes to Cells.14(6):727-737.doi:10.1111/j.1365-2443.2009.01305.x.
Topell S.,Hennecke J.,Glockshuber R.1999.Circularly permuted variants of the green fluorescent protein.FEBS Letters.457(2):283-289.doi:10.1016/s0014-5793(99)01044-3.
Topping J.F.1998.Tobacco transformation.In:Plant Virology Protocols:Springer.p.365-372.doi:10.1385/0-89603-385-6:365.
Tsien R.Y.2006.Breeding and building molecules to spy on cells and tumors.The Keio Journal of Medicine.55(4):127-140.doi:10.2302/kjm.55.127.
Yang P.C.Mahmood T.,2012.Western blot:Technique,theory,and trouble shooting.North American Journal of Medical Sciences.4(9):429.doi:10.4103/1947-2714.100998.