Npu DnaE内含肽构建及其对293T细胞高效反式剪接活性分析
|
摘要
目的构建Npu DnaE内含肽,探讨Npu DnaE内含肽是否在293T细胞中具备高效反式剪接活性。方法制备融合基因Vn-NDn-myc和NDc-Vc,插入pCDH-CMV-MCS-EF1-Puro载体,构建质粒。转染293T细胞,荧光显微镜下观察目的蛋白Venus是否形成。48 h后收集细胞蛋白,应用Western blot技术进一步印证。结果构建的质粒经限制性内切酶鉴定及测序比对正确,转染293T后可见共转染细胞组出现明亮荧光,弥漫分布于细胞质中,Western blot证明高量目的蛋白Venus形成。结论成功构建Npu DnaE内含肽,其能在293T细胞中发挥反式剪接的生物学功能,并具有高效的剪接效率及功能目的蛋白形成率。
Objective To construct Npu DnaE intein and to prove that Npu DnaE have highly efficient protein transsplicing activity in 293T cells. Methods We inserted the prepared fusion gene Vn-NDn-myc and NDc-Vc into pCDHCMV-MCS-EF1-Puro vector to construct the plasmid,then the plasmid was transfected into 293T cells,after that we observed the target protein to find the product of Venus with fluorescence microscope. We collected the protein after 48 hours and further proved the Venus protein by using Western blot. Results The recombinant plasmid was verified by enzyme digestion and sequence analysis. Fluorescence microscope showed that the fluorescence appeared in the co-transfected group and distributed all over the cytoplasm after transfection and the protein was highly produced in 293T cells,which was confirmed by Western blot. Conclusion Npu DnaE intein can play the biological functions of trans-splicing in 293T cells and with highly efficient protein trans-splicing activity and formation rate of the function target protein.
Objective To construct Npu DnaE intein and to prove that Npu DnaE have highly efficient protein transsplicing activity in 293T cells. Methods We inserted the prepared fusion gene Vn-NDn-myc and NDc-Vc into pCDHCMV-MCS-EF1-Puro vector to construct the plasmid,then the plasmid was transfected into 293T cells,after that we observed the target protein to find the product of Venus with fluorescence microscope. We collected the protein after 48 hours and further proved the Venus protein by using Western blot. Results The recombinant plasmid was verified by enzyme digestion and sequence analysis. Fluorescence microscope showed that the fluorescence appeared in the co-transfected group and distributed all over the cytoplasm after transfection and the protein was highly produced in 293T cells,which was confirmed by Western blot. Conclusion Npu DnaE intein can play the biological functions of trans-splicing in 293T cells and with highly efficient protein trans-splicing activity and formation rate of the function target protein.
引文
[1]Volkmann G,Iwai H.Protein trans-splicing and its use in structural biology:opportunities and limitations[J].Mol Biosyst,2010,6(11):2110-2121.
[2]Shi J,Muir TW.Development of a tandem protein trans-splicing system based on native and engineered split inteins[J].J Am Chem Soc,2005,127(17):6198-6206.
[3]Martin DD,Xu MQ,Evans TC.Characterization of a naturally occurring trans-splicingintein from Synechocystis sp.PCC6803[J].Biochemistry,2001,40(5):1393-1402.
[4]Hackenberger CP,Schwarzer D.Chemoselective ligation and modification strategies for peptides and proteins[J].Angew Chem Int Ed Engl,2008,47(52):10030-10074.
[5]Kent SB.Total chemical synthesis of proteins[J].Chem Soc Rev,2009,38(2):338-351.
[6]Fong BA,Wu WY,Wood DW.The potential role of self-cleaving purification tags in commercial-scale processes[J].Trends Biotech nol,2010,28(5):272-279.
[7]Vila-Perello M,Muir TW.Biological applications of protein splicing[J].Cell,2010,143(2):191-200.
[8]Flavell RR,Muir TW.Expressed protein ligation(EPL)in the study of signal transduction,ion conduction,and chromatin biology[J].Acc Chem Res,2009,42(1):107-116.
[9]Ozawa T,Sako Y,Sato M,et al.A genetic approach to identifying mitochondrial proteins[J].Nat Biotechnol,2003,21(3):287-293.
[10]Kanno A,Yamanaka Y,Hirano H,et al.Cyclic luciferase for real-time sensing of caspase-3 activities in living mammals[J].Angew Chem Int Ed Engl,2007,46(40):7595-7599.
[11]Zettler J,Schutz V,Mootz HD.The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction[J].FEBS Lett,2009,583(5):909-914.
[12]Iwai H,Zuger S,Jin J,et al.Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme[J].FEBS Lett,2006,580(7):1853-1858.
[13]Otomo T,Teruya K,Uegaki K,et al.Improved segmental isotope labeling of proteins and application to a larger protein[J].J Biomol NMR,1999,14(2):105-114.
[14]Cheriyan M,Pedamallu CS,Tori K,et al.Faster protein splicing with the Nostoc punctiforme DnaE intein using non-native extein residues[J].J Biol Chem,2013,288(9):6202-6211.
[15]Ramirez M,Valdes N,Guan D,et al.Engineering split intein DnaE from Nostoc punctiforme for rapid protein purification[J].Protein Eng Des Sel,2013,26(3):215-223.
[2]Shi J,Muir TW.Development of a tandem protein trans-splicing system based on native and engineered split inteins[J].J Am Chem Soc,2005,127(17):6198-6206.
[3]Martin DD,Xu MQ,Evans TC.Characterization of a naturally occurring trans-splicingintein from Synechocystis sp.PCC6803[J].Biochemistry,2001,40(5):1393-1402.
[4]Hackenberger CP,Schwarzer D.Chemoselective ligation and modification strategies for peptides and proteins[J].Angew Chem Int Ed Engl,2008,47(52):10030-10074.
[5]Kent SB.Total chemical synthesis of proteins[J].Chem Soc Rev,2009,38(2):338-351.
[6]Fong BA,Wu WY,Wood DW.The potential role of self-cleaving purification tags in commercial-scale processes[J].Trends Biotech nol,2010,28(5):272-279.
[7]Vila-Perello M,Muir TW.Biological applications of protein splicing[J].Cell,2010,143(2):191-200.
[8]Flavell RR,Muir TW.Expressed protein ligation(EPL)in the study of signal transduction,ion conduction,and chromatin biology[J].Acc Chem Res,2009,42(1):107-116.
[9]Ozawa T,Sako Y,Sato M,et al.A genetic approach to identifying mitochondrial proteins[J].Nat Biotechnol,2003,21(3):287-293.
[10]Kanno A,Yamanaka Y,Hirano H,et al.Cyclic luciferase for real-time sensing of caspase-3 activities in living mammals[J].Angew Chem Int Ed Engl,2007,46(40):7595-7599.
[11]Zettler J,Schutz V,Mootz HD.The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction[J].FEBS Lett,2009,583(5):909-914.
[12]Iwai H,Zuger S,Jin J,et al.Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme[J].FEBS Lett,2006,580(7):1853-1858.
[13]Otomo T,Teruya K,Uegaki K,et al.Improved segmental isotope labeling of proteins and application to a larger protein[J].J Biomol NMR,1999,14(2):105-114.
[14]Cheriyan M,Pedamallu CS,Tori K,et al.Faster protein splicing with the Nostoc punctiforme DnaE intein using non-native extein residues[J].J Biol Chem,2013,288(9):6202-6211.
[15]Ramirez M,Valdes N,Guan D,et al.Engineering split intein DnaE from Nostoc punctiforme for rapid protein purification[J].Protein Eng Des Sel,2013,26(3):215-223.