在大肠杆菌中高效表达可用于检测人源Rap1活性RapBD蛋白方法的建立
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摘要
【背景】Rap1是一种小GTP酶,其活性的检测方法少,目前主要依赖试剂盒,检测成本太高。而Rap1下游效应蛋白RalGDS具有Rap1结合结构域(Rap binding domain,RapBD),该结构域能与有活性的GTP-Rap1特异性结合。【目的】利用大肠杆菌外源表达GST-RapBD融合蛋白,建立经济的检测人源Rap1活性的方法。【方法】合成RapBD基因序列,插入pGEX-4T-1载体,使该质粒表达GST-RapBD融合蛋白,再利用GST亲和树脂结合大肠杆菌中表达的GST-RapBD融合蛋白,最后利用GST-RapBD融合蛋白Pulldown检测GTP-Rap1。【结果】建立了检测人源Rap1活性的方法。【结论】序列优化使得pGEX-4T-1载体在大肠杆菌中高效表达能特异性结合人源GTP-Rap1且带有GST标签的RapBD蛋白,提高了Pulldown实验检测GTP-Rap1的效率,降低了检测人源小G蛋白Rap1活性的成本。
[Background] Rap1 is a kind of small GTPase, and the method of its activity detection is very scanty. At present, the method mainly depends on commercialized kit, resulting in the high cost. RalGDS has the Rap binding domain(RapBD), which can bind to GTP-Rap1 specifically. [Objective] Establish an inexpensive method of detecting human Rap1 activity by exogenous GST-RapBD fusion protein expressed in Escherichia coli. [Methods] We constructed the plasmid with pGEX-4 T-1 vector expressing GST-RapBD fusion protein in E. coli, and then combined GST-RapBD with GST affinity resin. Finally, we used GST Pulldown assay to detect Rap1 activity. [Results] The method of detecting human Rap1 activity was established successfully. [Conclusion] Sequence optimization made pGEX-4 T-1 highly express the GST tagged RapBD protein in E. coli, which increased the efficiency and decreased the cost of Pulldown assay to detect GTP-Rap1.
[Background] Rap1 is a kind of small GTPase, and the method of its activity detection is very scanty. At present, the method mainly depends on commercialized kit, resulting in the high cost. RalGDS has the Rap binding domain(RapBD), which can bind to GTP-Rap1 specifically. [Objective] Establish an inexpensive method of detecting human Rap1 activity by exogenous GST-RapBD fusion protein expressed in Escherichia coli. [Methods] We constructed the plasmid with pGEX-4 T-1 vector expressing GST-RapBD fusion protein in E. coli, and then combined GST-RapBD with GST affinity resin. Finally, we used GST Pulldown assay to detect Rap1 activity. [Results] The method of detecting human Rap1 activity was established successfully. [Conclusion] Sequence optimization made pGEX-4 T-1 highly express the GST tagged RapBD protein in E. coli, which increased the efficiency and decreased the cost of Pulldown assay to detect GTP-Rap1.
引文
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[7]Parker WH,Qu ZC,May JM.Intracellular ascorbate prevents endothelial barrier permeabilization by thrombin[J].The Journal of Biological Chemistry,2015,290(35):21486-21497
[8]Xiong YW,Ye CJ,Yang NQ,et al.Ubc9 binds to ADAP and is required for Rap1 membrane recruitment,Rac1 activation,and integrin-mediated T cell adhesion[J].Journal of Immunology,2017,199(12):4142-4154
[9]Yang ZK,Kirton HM,Al-Owais M,et al.Epac2-Rap1 signaling regulates reactive oxygen species production and susceptibility to cardiac arrhythmias[J].Antioxidants&Redox Signaling,2017,27(3):117-132
[10]Genova T,Grolez GP,Camillo C,et al.TRPM8 inhibits endothelial cell migration via a non-channel function by trapping the small GTPase Rap1[J].The Journal of Cell Biology2017,216(7):2107-2130
[11]Zeng T,Cui ZM,Jiang W,et al.Optimization of the method for measurement of the activity of small GTP binding protein RhoA[J].Journal of Biology,2016,33(4):103-106(in Chinese)曾婷,崔照盟,蒋维,等.小G蛋白RhoA活性检测方法的优化[J].生物学杂志,2016,33(4):103-106
[12]Caron E,Self AJ,Hall A.The GTPase Rap1 controls functional activation of macrophage integrinαMβ2 by LPS and other inflammatory mediators[J].Current Biology,2000,10(16):974-978
[13]Chung J,Serezani CH,Huang SK,et al.Rap1 activation is required for Fcγreceptor-dependent phagocytosis[J].The Journal of Immunology,2008,181(8):5501-5509
[14]Wu AR,Chen H,Xu CF,et al.miR-203a is involved in HBx-induced inflammation by targeting Rap1a[J].Experimental Cell Research,2016,349(1):191-197
[15]Pizon V,Lerosey I,Chardin P,et al.Nucleotide sequence of a human cDNA encoding a ras-related protein(rap1B)[J].Nucleic Acids Research,1988,16(15):7719
[16]Minato N,Hattori M.Spa-1(Sipa1)and Rap signaling in leukemia and cancer metastasis[J].Cancer Science,2009,100(1):17-23
[17]Li SS,Guo XX,An S,et al.Biological function of the small Gprotein rap[J].Progress in Physiological Sciences,2016,47(1):14-20(in Chinese)李珊珊,郭晓汐,安输,等.小G蛋白Rap的信号通路与生物学功能[J].生理科学进展,2016,47(1):14-20
[18]Pizon V,Desjardins M,Bucci C,et al.Association of Rap1a and Rap1b proteins with late endocytic/phagocytic compartments and Rap2a with the Golgi complex[J].Journal of Cell Science,1994,107:1661-1670
[19]Kawasaki H,Springett GM,Mochizuki N,et al.A family of cAMP-binding proteins that directly activate Rap1[J].Science,1998,282(5397):2275-2279
[2]Katagiri K,Maeda A,Shimonaka M,et al.RAPL,a Rap1-binding molecule that mediates Rap1-induced adhesion through spatial regulation of LFA-1[J].Nature Immunology,2003,4(8):741-748
[3]Jeong HW,Li ZG,Brown MD,et al.IQGAP1 binds Rap1 and modulates its activity[J].The Journal of Biological Chemistry,2007,282(28):20752-20762
[4]Vigil D,Cherfils J,Rossman KL,et al.Ras superfamily GEFs and GAPs:validated and tractable targets for cancer therapy?[J]Nature Reviews Cancer,2010,10(12):842-857
[5]Raaijmakers JH,Bos JL.Specificity in Ras and Rap signaling[J].The Journal of Biological Chemistry,2009,284(17):10995-10999
[6]Spaargaren M,Bischoff JR.Identification of the guanine nucleotide dissociation stimulator for Ral as a putative effector molecule of R-ras,H-ras,K-ras,and Rap[J].Proceedings of the National Academy of Sciences of the United States of America,1994,91(26):12609-12613
[7]Parker WH,Qu ZC,May JM.Intracellular ascorbate prevents endothelial barrier permeabilization by thrombin[J].The Journal of Biological Chemistry,2015,290(35):21486-21497
[8]Xiong YW,Ye CJ,Yang NQ,et al.Ubc9 binds to ADAP and is required for Rap1 membrane recruitment,Rac1 activation,and integrin-mediated T cell adhesion[J].Journal of Immunology,2017,199(12):4142-4154
[9]Yang ZK,Kirton HM,Al-Owais M,et al.Epac2-Rap1 signaling regulates reactive oxygen species production and susceptibility to cardiac arrhythmias[J].Antioxidants&Redox Signaling,2017,27(3):117-132
[10]Genova T,Grolez GP,Camillo C,et al.TRPM8 inhibits endothelial cell migration via a non-channel function by trapping the small GTPase Rap1[J].The Journal of Cell Biology2017,216(7):2107-2130
[11]Zeng T,Cui ZM,Jiang W,et al.Optimization of the method for measurement of the activity of small GTP binding protein RhoA[J].Journal of Biology,2016,33(4):103-106(in Chinese)曾婷,崔照盟,蒋维,等.小G蛋白RhoA活性检测方法的优化[J].生物学杂志,2016,33(4):103-106
[12]Caron E,Self AJ,Hall A.The GTPase Rap1 controls functional activation of macrophage integrinαMβ2 by LPS and other inflammatory mediators[J].Current Biology,2000,10(16):974-978
[13]Chung J,Serezani CH,Huang SK,et al.Rap1 activation is required for Fcγreceptor-dependent phagocytosis[J].The Journal of Immunology,2008,181(8):5501-5509
[14]Wu AR,Chen H,Xu CF,et al.miR-203a is involved in HBx-induced inflammation by targeting Rap1a[J].Experimental Cell Research,2016,349(1):191-197
[15]Pizon V,Lerosey I,Chardin P,et al.Nucleotide sequence of a human cDNA encoding a ras-related protein(rap1B)[J].Nucleic Acids Research,1988,16(15):7719
[16]Minato N,Hattori M.Spa-1(Sipa1)and Rap signaling in leukemia and cancer metastasis[J].Cancer Science,2009,100(1):17-23
[17]Li SS,Guo XX,An S,et al.Biological function of the small Gprotein rap[J].Progress in Physiological Sciences,2016,47(1):14-20(in Chinese)李珊珊,郭晓汐,安输,等.小G蛋白Rap的信号通路与生物学功能[J].生理科学进展,2016,47(1):14-20
[18]Pizon V,Desjardins M,Bucci C,et al.Association of Rap1a and Rap1b proteins with late endocytic/phagocytic compartments and Rap2a with the Golgi complex[J].Journal of Cell Science,1994,107:1661-1670
[19]Kawasaki H,Springett GM,Mochizuki N,et al.A family of cAMP-binding proteins that directly activate Rap1[J].Science,1998,282(5397):2275-2279