蛋白质酪氨酸磷酸酶MEG2的纯化表征及其多克隆抗体的制备
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摘要
为了研究PTP-MEG2的催化结构域在细胞信号转导中的功能,我们以含有PTP-MEG2全长基因的质粒为模板,克隆了PTP-MEG2催化结构域基因△MEG2,构建了pT7-△MEG2质粒,测序后结果正确。通过转化克隆宿主大肠杆菌DH5α进行扩增后,再转化表达宿主大肠杆菌Rosetta DE3,进行PTP-MEG2的高效表达,并利用正离子交换柱和负离子交换柱纯化该蛋白质,得到了纯化后的PTP-MEG2蛋白,并具有较高的比活。再对纯化后的PTP-MEG2蛋白进行表征,得到其酶促反应的最适温度,最适离子强度,最适PH值。对PTP-MEG2蛋白的稳定性进行研究,测得其Km值。利用纯化后的蛋白对家兔进行免疫,来制备PTP-MEG2的多克隆抗体,并利用“PVDF固定抗原亲和层析法”对其多克隆抗体进行纯化,以期得到纯度、效价及灵敏度较高的PTP-MEG2抗体,为以后对其在细胞中的生理功能和组织分布的研究打下良好的基础
MEG2 protein tyrosine phosphatase (PTP-MEG2), which is PTP megacaryocyte1 protein tyrosine kinase (PTP) subfamily members of the family also includes PTPH1, PTPBAS and PTPD1.Its open reading frame encoding a 593 amino acids and contains molecular weight of 68 kDa protein,It did not clear signal and transmembrane sequence, PTP for its C-terminal catalytic domain, and other known PTP is 30-40% homology;N-terminal 250 amino acids, and the cells within the retinal uncoupling protein 28% homology with the SEC 14p yeast protein with 24% identity,Yeast SEC14p protein phosphatidylinositol transfer activity and is adopted by the yeast Golgi protein secretion of the necessary role of the protein, which may be related to the Gorky PTP-MEG2 of the function.PTP-MEG2 reorganization of the protein can be expressed in E. coli and soluble PTP vitality.PTP-MEG2 mRNA in the different organs of the human body from 12 kinds of cells can be detected, it also shows that such a broad expression of PTP.PTP-MEG2 hinted that the structure of this enzyme may be involved in the tyrosine phosphorylation of the hydrophobic ligand and the transfer of the functions of Gorky.
Of polycythemia vera (PV) is a myeloproliferative disease, accompanied by red blood cells, myeloid blood cells and the excessive proliferation.Previously, confirmed from the experimental PV patients erythroid colony-forming cells (ECFC) containing an activity over the coupling membrane tyrosine phosphatase now confirm that this enzyme is PTP-MEG2, with a structure containing lipid within the domain of the cell.PTP-MEG2 in the PV cells because of its excessive activation of the membrane fraction on the increase in the distribution of the.With the development of a mature ECFC RBC, PTP-MEG2 protein level is gradually decreasing, but the coupling membrane PTP-MEG2 in PV cells has lasted a long time, and enhanced cell colony-forming ability.PTP-MEG2 dominant negative mutant expression in vitro in both inhibited the growth and expansion of ECFC. This shows that PTP-MEG2 erythroid cells in the development process plays an important role, for the development of drugs to reduce the proliferation of red blood cells provides a good therapeutic targets.
Main results are as follows:
1. PTP-MEG2 catalytic domain of the gene cloning and expression of△MEG2.
We use pBluescript II KS MEG2 as a template, which was amplified by PCR, and shert its N-terminal and C-terminal into EcoRI restriction endonuclease sites and the suspension of codon, DNA electrophoresis results shows that the PCR product sizes are correct. PCR amplification will be the PTP-MEG2 gene, and we connect it into pT7 expression vector, and then digested with EcoR I, the DNA electrophoresis identification shows the correct size. Through recombinant DNA sequencing proved the correct sequence, recombinant DNA expression vector will be amplified into E. coli Rosetta DE3, access to the strain and high expression of a highly efficient expression.
2. Purification and Characterization of PTP-MEG2
(1): Extract of target protein extract: Put the bacterium in the centrifuge tube and at the speed of 5000 rpm centrifugal 12 min, disposable medium, add 20 ml PBS and dissolved after ultrasonic broken blender 5 min, turn into the tube Centrifugal at the speed of 2000 rpm for 5 min to remove cell debris, and 15000 rpm centrifugal 12 min, disposable supernatant obtained precipitation for the bacterial extracts. Will be induced before and after induction of cell protein precipitation transferred to the same concentration for the entire electrophoretic analysis whether the large number of protein was expressed.
(2): PTP-MEG2 Purification: Extract the Q-Sepharose Fast Flow ion-exchange column chromatography and SP-Sephadex ion-exchange column chromatography by the purified protein. Electrophoretic analysis purity above 95%.
(3): PTP-MEG2 Characterization: Configurate different pH and ionic strength of the buffer, protein determination in different pH and ionic strength and temperature under the condition of enzymatic reaction. By its enzymatic reaction optimum conditions.
3. PTP -MEG2 polyclonal antibody preparation and purification
We use the purified PTP-MEG2 immune rabbit, sera obtained through PVDF fixed antigen affinity chromatography column and purified from the anti PTP-MEG2 polyclonal antibody. The ECL detection, purified antibody titer after 1:10000 can be reached (V/V); the sensitivite of antigen-antibody is 1 ng. The purified antibody titer can be reached 1:2500 (V/V); sensitivity antigen-antibody is 0.1 ng. Thus, the purified antibody, antigen sensitivite increased by 10 times; through the entire electrophoresis and Weston Blot Detection of antibody purity of more than 90% can be achieved, it is in the line with experimental immunization requirements. Purified antibody serum after 56℃, 30 min after heating inactivated by adding final concentration of 1/1000 of sodium azide, cryopreservation in -80℃.
MEG2 protein tyrosine phosphatase (PTP-MEG2), which is PTP megacaryocyte1 protein tyrosine kinase (PTP) subfamily members of the family also includes PTPH1, PTPBAS and PTPD1.Its open reading frame encoding a 593 amino acids and contains molecular weight of 68 kDa protein,It did not clear signal and transmembrane sequence, PTP for its C-terminal catalytic domain, and other known PTP is 30-40% homology;N-terminal 250 amino acids, and the cells within the retinal uncoupling protein 28% homology with the SEC 14p yeast protein with 24% identity,Yeast SEC14p protein phosphatidylinositol transfer activity and is adopted by the yeast Golgi protein secretion of the necessary role of the protein, which may be related to the Gorky PTP-MEG2 of the function.PTP-MEG2 reorganization of the protein can be expressed in E. coli and soluble PTP vitality.PTP-MEG2 mRNA in the different organs of the human body from 12 kinds of cells can be detected, it also shows that such a broad expression of PTP.PTP-MEG2 hinted that the structure of this enzyme may be involved in the tyrosine phosphorylation of the hydrophobic ligand and the transfer of the functions of Gorky.
Of polycythemia vera (PV) is a myeloproliferative disease, accompanied by red blood cells, myeloid blood cells and the excessive proliferation.Previously, confirmed from the experimental PV patients erythroid colony-forming cells (ECFC) containing an activity over the coupling membrane tyrosine phosphatase now confirm that this enzyme is PTP-MEG2, with a structure containing lipid within the domain of the cell.PTP-MEG2 in the PV cells because of its excessive activation of the membrane fraction on the increase in the distribution of the.With the development of a mature ECFC RBC, PTP-MEG2 protein level is gradually decreasing, but the coupling membrane PTP-MEG2 in PV cells has lasted a long time, and enhanced cell colony-forming ability.PTP-MEG2 dominant negative mutant expression in vitro in both inhibited the growth and expansion of ECFC. This shows that PTP-MEG2 erythroid cells in the development process plays an important role, for the development of drugs to reduce the proliferation of red blood cells provides a good therapeutic targets.
Main results are as follows:
1. PTP-MEG2 catalytic domain of the gene cloning and expression of△MEG2.
We use pBluescript II KS MEG2 as a template, which was amplified by PCR, and shert its N-terminal and C-terminal into EcoRI restriction endonuclease sites and the suspension of codon, DNA electrophoresis results shows that the PCR product sizes are correct. PCR amplification will be the PTP-MEG2 gene, and we connect it into pT7 expression vector, and then digested with EcoR I, the DNA electrophoresis identification shows the correct size. Through recombinant DNA sequencing proved the correct sequence, recombinant DNA expression vector will be amplified into E. coli Rosetta DE3, access to the strain and high expression of a highly efficient expression.
2. Purification and Characterization of PTP-MEG2
(1): Extract of target protein extract: Put the bacterium in the centrifuge tube and at the speed of 5000 rpm centrifugal 12 min, disposable medium, add 20 ml PBS and dissolved after ultrasonic broken blender 5 min, turn into the tube Centrifugal at the speed of 2000 rpm for 5 min to remove cell debris, and 15000 rpm centrifugal 12 min, disposable supernatant obtained precipitation for the bacterial extracts. Will be induced before and after induction of cell protein precipitation transferred to the same concentration for the entire electrophoretic analysis whether the large number of protein was expressed.
(2): PTP-MEG2 Purification: Extract the Q-Sepharose Fast Flow ion-exchange column chromatography and SP-Sephadex ion-exchange column chromatography by the purified protein. Electrophoretic analysis purity above 95%.
(3): PTP-MEG2 Characterization: Configurate different pH and ionic strength of the buffer, protein determination in different pH and ionic strength and temperature under the condition of enzymatic reaction. By its enzymatic reaction optimum conditions.
3. PTP -MEG2 polyclonal antibody preparation and purification
We use the purified PTP-MEG2 immune rabbit, sera obtained through PVDF fixed antigen affinity chromatography column and purified from the anti PTP-MEG2 polyclonal antibody. The ECL detection, purified antibody titer after 1:10000 can be reached (V/V); the sensitivite of antigen-antibody is 1 ng. The purified antibody titer can be reached 1:2500 (V/V); sensitivity antigen-antibody is 0.1 ng. Thus, the purified antibody, antigen sensitivite increased by 10 times; through the entire electrophoresis and Weston Blot Detection of antibody purity of more than 90% can be achieved, it is in the line with experimental immunization requirements. Purified antibody serum after 56℃, 30 min after heating inactivated by adding final concentration of 1/1000 of sodium azide, cryopreservation in -80℃.
引文
[1] 孙大业,郭艳林主编。细胞信号转导 ,科学出版社,2001.
[2] Lienard H, Bruhns P, Malbec O, Fridman WH, Daeron M. Signal cell activation. regulatory priteins negatively regulate immuneoreceptor-dependent。J Biol Chem, 1999;274:32493-32498.
[3] Cantley LC, Anger KR, Carpenter C, Duckworth B, Graziani A, Kapeller R, Soltoff S. Oncogene and signal transduction. Cell,1991;64:281-302.
[4] Houshino R, Chatani Y, Yamori T, Tsuruo T, Oka H, Yashida O, Shimada Y, Arii S, Wada H, Fujimoto J, Kohno M. Constitutiveactivation of the 41-143-kDa mitogen-activated protein kinase signaling pathway in human tumor. Oncogene, 1999;18:813-822.
[5] Sivaraman VS, Wang H, Nuovo GJ, Malbon CC. Hyperexpression of mitogen-activated protein kinase in human breast cancer. J Clin Invest, 1997;99:1478-1483.
[6] 成军,陈菊梅. 丙型肝炎病毒核心蛋白结合蛋白的研究进展.国外医学病毒学分册 2000;7:123-127.
[7] Schindler CW. Series introduction. JAK-STAT signaling in human disease. J Clin Invest 2002;109:1133-1137.
[8] O'Shea JJ,Gadina M,Schreiber RD. Cytokine signaling in 2002: new surprises in the Jak/Stat pathway. Cell, 2002;109 ( Suppl ):S121-131.
[9] Yang, J. et al. Identification of a family of calcium sensors as protein ligands of inositol trisphosphate receptor Ca2+ release channels. Proc. Natl Acad. Sci. USA ,1999,99: 7711-7716.
[10] Itoh, Y. et al. Free fatty acids regulate insulin secretion from pancreatic cells through GPR40. Nature 2000,422:173-176.
[11] Fuqua SA, Fitzgerald SD, Allred DC, et al. Inhibition of estrogen receptor action by a naturally occurring variant in human breast tumors. Cancer Res, 1992;52, 483-486.
[12] Krebs EG. Protein phosphorylatin and cellular regulation I (Novel Lecture) Angew Chem 1993, 32(8): 1122–1129.
[13] Fischer EH. Protein phosphorylatin and cellular regulation II (Novel Lecture) Angew Chem 1993, 32(8): 1130–1137.
[14] Hunter T. Signaling--2000 and beyond. Cell, 2000, 100: 113–127。
[15] Venter JC Adams MD, Myerset EW et al. The Sequence of the Human Genome. Science 2001, 291: 1304–1351.
[16] Lander ES et al. Initial sequencing and analysis of the human genome Nature 2001, 409: 860–921.
[17] Streuli M. Protein tyrosine phosphatases in signaling. Curr Opinion in Cell Biol 1996 183:182-188.
[18] Tonks NK, Diltz DC, and Fischer EH. Purification of the major protein tyrosine phosphatases. J Biol Chem 1988, 263(14): 6722–6725.
[19] Fischer EH, Charbonneau H, Tonk NK. Protein tyrosine phophatases:a diverse family of intracellular and transmembrane enzymes. Science 1991, 253: 401–406.
[20] Robinson DR, Yi-Mi Wu, Su-Fang Lin. The protein tyrosine kinase family of the human genome. Oncogene 2000, 19: 5548–5557.
[21] Walton KM, Dixon JE. Protein tyrosine phosphatases. Ann Rev Biochem 1993, 62: 101–120.
[22] Fauman EB and Saper MA. Structure and function of the protein tyrosine phosphatases. Trends Biochem Sci 1996, 21: 413-417.
[23] Z.Y. Zhang, Y. Wang, Dixon JE. Dissecting the catalytic mechanism of protein-tyrosine phosphatases. Proc Natl Acad Sci USA 1994, 91: 1624–1627.
[24] 翟中和等主编。 细胞生物学 第一版 高等教育出版社 ,2000.
[25] Minxiang Gu and Philip W. Majerus . The Properties of the Protein Tyrosine Phosphatase PTPMEG J Biol Chem 1996 ,pp. 27751–27759.
[26] MINXIANG GU, KUN MENG, AND PHILIP W. MAJERUS The effect of overexpression of the protein tyrosine phosphatase PTPMEG on cell growth and on colony formation in soft agar in COS-7 cells Cell Biology 1996,pp. 12980–12985.
[27] Katsunori Hironaka?, Hisashi Umemori?, Tohru Tezuka?, Masayoshi Mishina§, and Tadashi Yamamoto. The Protein-tyrosine Phosphatase PTPMEG Interacts with Glutamate Receptor d2 and e Subunits J Biol Chem 2000 ,pp. 16167–16173.
[28] Sambrook J, Russell DW. 分子克隆实验指南 第三版,黄培堂等译。科学出版社,2002.
[29] 卢胜栋主编。现代分子生物学技术 高等教育出版社,1993.
[2] Lienard H, Bruhns P, Malbec O, Fridman WH, Daeron M. Signal cell activation. regulatory priteins negatively regulate immuneoreceptor-dependent。J Biol Chem, 1999;274:32493-32498.
[3] Cantley LC, Anger KR, Carpenter C, Duckworth B, Graziani A, Kapeller R, Soltoff S. Oncogene and signal transduction. Cell,1991;64:281-302.
[4] Houshino R, Chatani Y, Yamori T, Tsuruo T, Oka H, Yashida O, Shimada Y, Arii S, Wada H, Fujimoto J, Kohno M. Constitutiveactivation of the 41-143-kDa mitogen-activated protein kinase signaling pathway in human tumor. Oncogene, 1999;18:813-822.
[5] Sivaraman VS, Wang H, Nuovo GJ, Malbon CC. Hyperexpression of mitogen-activated protein kinase in human breast cancer. J Clin Invest, 1997;99:1478-1483.
[6] 成军,陈菊梅. 丙型肝炎病毒核心蛋白结合蛋白的研究进展.国外医学病毒学分册 2000;7:123-127.
[7] Schindler CW. Series introduction. JAK-STAT signaling in human disease. J Clin Invest 2002;109:1133-1137.
[8] O'Shea JJ,Gadina M,Schreiber RD. Cytokine signaling in 2002: new surprises in the Jak/Stat pathway. Cell, 2002;109 ( Suppl ):S121-131.
[9] Yang, J. et al. Identification of a family of calcium sensors as protein ligands of inositol trisphosphate receptor Ca2+ release channels. Proc. Natl Acad. Sci. USA ,1999,99: 7711-7716.
[10] Itoh, Y. et al. Free fatty acids regulate insulin secretion from pancreatic cells through GPR40. Nature 2000,422:173-176.
[11] Fuqua SA, Fitzgerald SD, Allred DC, et al. Inhibition of estrogen receptor action by a naturally occurring variant in human breast tumors. Cancer Res, 1992;52, 483-486.
[12] Krebs EG. Protein phosphorylatin and cellular regulation I (Novel Lecture) Angew Chem 1993, 32(8): 1122–1129.
[13] Fischer EH. Protein phosphorylatin and cellular regulation II (Novel Lecture) Angew Chem 1993, 32(8): 1130–1137.
[14] Hunter T. Signaling--2000 and beyond. Cell, 2000, 100: 113–127。
[15] Venter JC Adams MD, Myerset EW et al. The Sequence of the Human Genome. Science 2001, 291: 1304–1351.
[16] Lander ES et al. Initial sequencing and analysis of the human genome Nature 2001, 409: 860–921.
[17] Streuli M. Protein tyrosine phosphatases in signaling. Curr Opinion in Cell Biol 1996 183:182-188.
[18] Tonks NK, Diltz DC, and Fischer EH. Purification of the major protein tyrosine phosphatases. J Biol Chem 1988, 263(14): 6722–6725.
[19] Fischer EH, Charbonneau H, Tonk NK. Protein tyrosine phophatases:a diverse family of intracellular and transmembrane enzymes. Science 1991, 253: 401–406.
[20] Robinson DR, Yi-Mi Wu, Su-Fang Lin. The protein tyrosine kinase family of the human genome. Oncogene 2000, 19: 5548–5557.
[21] Walton KM, Dixon JE. Protein tyrosine phosphatases. Ann Rev Biochem 1993, 62: 101–120.
[22] Fauman EB and Saper MA. Structure and function of the protein tyrosine phosphatases. Trends Biochem Sci 1996, 21: 413-417.
[23] Z.Y. Zhang, Y. Wang, Dixon JE. Dissecting the catalytic mechanism of protein-tyrosine phosphatases. Proc Natl Acad Sci USA 1994, 91: 1624–1627.
[24] 翟中和等主编。 细胞生物学 第一版 高等教育出版社 ,2000.
[25] Minxiang Gu and Philip W. Majerus . The Properties of the Protein Tyrosine Phosphatase PTPMEG J Biol Chem 1996 ,pp. 27751–27759.
[26] MINXIANG GU, KUN MENG, AND PHILIP W. MAJERUS The effect of overexpression of the protein tyrosine phosphatase PTPMEG on cell growth and on colony formation in soft agar in COS-7 cells Cell Biology 1996,pp. 12980–12985.
[27] Katsunori Hironaka?, Hisashi Umemori?, Tohru Tezuka?, Masayoshi Mishina§, and Tadashi Yamamoto. The Protein-tyrosine Phosphatase PTPMEG Interacts with Glutamate Receptor d2 and e Subunits J Biol Chem 2000 ,pp. 16167–16173.
[28] Sambrook J, Russell DW. 分子克隆实验指南 第三版,黄培堂等译。科学出版社,2002.
[29] 卢胜栋主编。现代分子生物学技术 高等教育出版社,1993.