烟草NtGCN2的原核表达、纯化及多克隆抗体制备
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摘要
GCN2 (general control non-derepressible-2)磷酸化真核翻译起始因子(eukaryotic translation initiation factor 2α, eIF2α)而抑制蛋白的翻译,从而感知氨基酸的缺乏并对各种胁迫做出响应。为了深入研究烟草(Nicotiana tabacum) NtGCN2的功能,本实验构建了烟草NtGCN2全长和N端NtGCN2(1～437 aa)的原核表达载体pET15b-NtGCN2和pCzn1-NtGCN2(1～437 aa),分别在大肠杆菌(Escherichia coli) BL21-CodonPlus-(DE3)-RIPL、BL21-CodonPlus-(DE3)-RPL和BL21(DE3) Plys进行原核表达。Western blot结果显示,烟草NtGCN2全长和NtGCN2(1～437 aa)蛋白分别在大肠杆菌BL21-CodonPlus-(DE3)-RIPL和BL21(DE3) Plys中成功表达。其次,对NtGCN2全长蛋白的表达条件进行优化。结果显示,在16℃、0.5 mmol/L异丙基硫代半乳糖苷(isopropylβ-D-thiogalactoside,IPTG)、13 h的诱导条件下,BL21-CodonPlus-(DE3)-RIPL的上清中获得了可溶的NtGCN2蛋白。采用阴离子交换柱Hitrap Q和Ni2+-NTA琼脂糖亲和层析对蛋白进行初步纯化。采用包涵体复性的方法获得较纯的NtGCN2和NtGCN2(1～437 aa)蛋白。其中,NtGCN2(1～437 aa)通过Ni2+-NTA琼脂糖亲和层析纯化后获得条带单一的纯蛋白。最后,利用获得的重组蛋白制备抗体,并对原核表达的NtGCN2全长和NtGCN2(1～437 aa)蛋白进行Western blot检测,结果表明制备的抗体均能有效识别NtGCN2蛋白。本研究实现了NtGCN2蛋白的原核表达和纯化,制备了NtGCN2的多克隆抗体,为进一步探讨植物GCN2的功能提供了基础资料。
General control non-derepressible-2(GCN2) regulates protein synthesis by phosphorylation of eukaryotic translation initiation factor 2(eIF2α) and responding to amino acid deficiency and various stresses.At present, the action mechanism of GCN2 in yeast and animals has been relatively clear, but the regulation mechanism of plant GCN2 is not clear. In order to further study the function of GCN2, 2 special primer sequences were designed to amplify the full-length NtGCN2 and N-terminus(1～437 aa) gene sequences, and then inserted them to p ET-15 b and pCzn1 vectors, respectively. The prokaryotic expression vectors pET15bNtGCN2 and pCzn1-NtGCN2(1～437 aa) were transformed into Escherichia coli BL21-CodonPlus-(DE3)-RIPL, BL21-CodonPlus-(DE3)-RPL and BL21(DE3) Plys for expression, respectively. The full lengthNtGCN2 and N-terminal NtGCN2(1～437 aa) proteins were successfully expressed in E.coli BL21-CodonPlus-(DE3)-RIPL and BL21(DE3) Plys, respectively. The expressed protein were identified by Western blot using6 × His mouse antibody as a primary antibody. The prokaryotic expression condition of full-length NtGCN2 including induction concentration of isopropyl β-D-1-thiogalactopyranoside(IPTG), culture temperature and culture time were optimized. These results showed that soluble NtGCN2 protein was obtained in the supernatant of BL21-CodonPlus-(DE3)-RIPL under the conditions of 16 ℃, 0.5 mmol/L IPTG and 13 h induction. The protein was purified by affinity chromatography on anion exchange column Hitrap Q and Ni2+-NTA agarose and the NtGCN2 protein was not well separated and purified. Furthermore, the purified NtGCN2 and NtGCN2(1～437 aa) proteins were obtained by inclusion body renaturation, and a single band of NtGCN2(1～437 aa) pure protein was obtained by further Ni2+-NTA agarose affinity chromatography. Finally, The purified recombinant protein was used as immunized rabbit antigen and polyclonal antibody against NtGCN2 was successfully prepared, the titer and specificity of the antibodies were analyzed. Enzyme-linked immune sorbent assay(ELISA) assay showed that the antibodies titer reached over 1∶50 000. Western blot analysis showed that the prepared NtGCN2 full-length and NtGCN2(1～437 aa) polyclonal antibody could specifically recognize the recombined NtGCN2 protein. In this study, the prokaryotic expression and purification of NtGCN2 protein were achieved, and the antibody against NtGCN2 was prepared, which would provide data for the further exploration of the function of plant GCN2.
General control non-derepressible-2(GCN2) regulates protein synthesis by phosphorylation of eukaryotic translation initiation factor 2(eIF2α) and responding to amino acid deficiency and various stresses.At present, the action mechanism of GCN2 in yeast and animals has been relatively clear, but the regulation mechanism of plant GCN2 is not clear. In order to further study the function of GCN2, 2 special primer sequences were designed to amplify the full-length NtGCN2 and N-terminus(1～437 aa) gene sequences, and then inserted them to p ET-15 b and pCzn1 vectors, respectively. The prokaryotic expression vectors pET15bNtGCN2 and pCzn1-NtGCN2(1～437 aa) were transformed into Escherichia coli BL21-CodonPlus-(DE3)-RIPL, BL21-CodonPlus-(DE3)-RPL and BL21(DE3) Plys for expression, respectively. The full lengthNtGCN2 and N-terminal NtGCN2(1～437 aa) proteins were successfully expressed in E.coli BL21-CodonPlus-(DE3)-RIPL and BL21(DE3) Plys, respectively. The expressed protein were identified by Western blot using6 × His mouse antibody as a primary antibody. The prokaryotic expression condition of full-length NtGCN2 including induction concentration of isopropyl β-D-1-thiogalactopyranoside(IPTG), culture temperature and culture time were optimized. These results showed that soluble NtGCN2 protein was obtained in the supernatant of BL21-CodonPlus-(DE3)-RIPL under the conditions of 16 ℃, 0.5 mmol/L IPTG and 13 h induction. The protein was purified by affinity chromatography on anion exchange column Hitrap Q and Ni2+-NTA agarose and the NtGCN2 protein was not well separated and purified. Furthermore, the purified NtGCN2 and NtGCN2(1～437 aa) proteins were obtained by inclusion body renaturation, and a single band of NtGCN2(1～437 aa) pure protein was obtained by further Ni2+-NTA agarose affinity chromatography. Finally, The purified recombinant protein was used as immunized rabbit antigen and polyclonal antibody against NtGCN2 was successfully prepared, the titer and specificity of the antibodies were analyzed. Enzyme-linked immune sorbent assay(ELISA) assay showed that the antibodies titer reached over 1∶50 000. Western blot analysis showed that the prepared NtGCN2 full-length and NtGCN2(1～437 aa) polyclonal antibody could specifically recognize the recombined NtGCN2 protein. In this study, the prokaryotic expression and purification of NtGCN2 protein were achieved, and the antibody against NtGCN2 was prepared, which would provide data for the further exploration of the function of plant GCN2.
引文
陈德鑫,李雯雯,李思斌,等.2017.烟草NteIF2α的原核表达、纯化及多克隆抗体制备和应用[J].农业生物技术学报,25(1):50-57.(Chen D X,Li W W,Li S B,et al2017.Prokaryotic expression,purification of NteIF2αand preparation and application of polyclonal antibody in Nicotiana tabacum[J].Journal of Agricultural Biotechnology,25(1):50-57.)
陈浩,陈于红,朱德煦,等.2002.重组蛋白质纯化技术[J].中国生物工程杂志,22(5):87-92.(Chen H,Chen Y HZhu D X,et al.2002.Recombinant protein purification technology[J].China Bioengineering,22(5):87-92.)
陈明,陈文静.2011.几种微生物表达系统的比较[J].安徽农学通报,17(03):68-71.(Chen M,Chen W J.2011.Comparison of several microbial expression systems[J].Anhui Agricultural Science Bulletin,17(03):68-71.)
韩学波,许崇波,曾瑾.2006.大肠杆菌中外源基因的表达调节[J].生物技术通讯,17(03):418-420.(Han X B,Xu CB,Zeng J.2006.Expression regulation of foreign genes in E.coli[J].Letters in Biotechnology,17(03):418-420.)
何伟.2017.重组大肠杆菌表达包涵体药物蛋白的复性与纯化研究[D].硕士学位论文,华东理工大学,导师:王学东,pp.18-21.(He W.2017.Refolding and purification of recombinant E.coli expressing inclusion body drug proteins[D].Thesis for M.S.,East China University of Science and Technology,Supervisor:Wang X D,pp.18-21.)
黄传臻,刘香利,曹汝菲,等.2017.小麦CWI-B1的原核表达、纯化与多克隆抗体制备[J].农业生物技术学报,25(07):1102-1110.(Huang Z Z,Liu X L,Cao R F,et al.2017.Prokaryotic expression and purification of wheat CWI-B1 and preparation of polyclonal antibodies[J].Journal of Agricultural Biotechnology,25(07):1102-1110.)
卢晟晔,王丽颖.2006.大肠杆菌中外源蛋白高效表达的影响因素及策略研究的新进展[J].中国实验诊断学,10(09):1100-1103.(Lu S Y,Wang L Y.2006.New progress in influencing factors and strategies of overexpression of foreign proteins in E.coli[J].Chinese Journal of Laboratory Diagnosis,10(09):1100-1103.)
李厚华,孔伟胜,魏圆圆,等.2015.植物蛋白质翻译水平的调控[J].植物生理学报,51(2):159-165.(Li H H,Kong W S,Wei Y Y,et al.2015.Regulation of protein translation level in plants[J].Plant Physiology Journal,51(2):159-165.)
许文涛,芦云,罗云波,等.2008.潮霉素磷酸转移酶(hpt)基因的原核表达、蛋白纯化及其抗体制备[J].农业生物技术学报,16(4):647-652.(Xu W T,Lu Y,Luo Y B,et al.2008.Expression of hygromycin-β-phosphotransferase(hpt)gene in Escherichia coli,purification of HPTprotein and preparation of its polyclonal antibodies[J].Journal of Agricultural Biotechnology,16(4):647-652.)
张康旭,刘国顺,李雯雯,等.2014.烟草NtGCN2的克隆与表达分析[J].植物生理学报,50(9):1406-1412.(Zhang K X,Liu G S,Li W W,et al.2014.Cloning and expression analysi s of NtGCN2 in Nicotiana tabacum[J].Plant Physiology Journal,50(9):1406-1412.)
Cherkasova V A,Hinnebusch A G.2003.Translational control by TOR and TAP42 through dephosphorylation of eIF2alpha kinase GCN2[J].Genes Development,17(7):859-872.
Donnelly N,Gorman A M,Gupta S,et al.2013.The e IF2αkinases:Their structures and functions[J].Cellular and Molecular Life Sciences,70(19):3493-3511.
Garcia M A,Meurs E F,Esteban M.2007.The dsRNA protein kinase PKR:Virus and cell control[J].Biochimie,89(6-7):799-811.
Grallert B,Boye E.2013.GCN2,an old dog with new tricks[J].Biochemical Society Transactions,41(6):1687-1691
Halford N G.2006.Regulation of carbon and amino acid metabolism,roles of sucrose nonfermenting-1-related protein kinase-1 and general control nonderepressible-2-related protein kinase[J].Advance in Botanical Research43(05):93-142.
Hey S J,Byrne E,Halford N G.2010.The interface between metabolic and stress signaling[J].Annals of Botany,105(2):197-203.
Hinnebusch A G.2005.Translational regulation of GCN4 and the general amino acid control of yeast[J].Annual Review of Microbiology,59(1):407-450.
Kimball S R.1999.Eukaryotic initiation factor eIF2[J].International Journal of Biochemistry,31(1):25-29.
Lageix S,Rothenburg S,Dever T E,et al.2014.Enhanced interaction between pseudokinase and kinase domains in GCN2 stimulates eIF2αphosphory lation in starved cells[J].PLoS Genetics,10(5):e1004326.
Liu J,Cai C G,Guo Q,et al.2013.Secretory expression and efficient purification of recombinant anthrax toxin letha factor with full biological activity in E.coli.[J].Protein Expression and Purification,89(1):56-61.
Li M W,Auyeung W K,Lam H M.2013.The GCN2 homologue in Arabidopsis thaliana interacts with uncharged tRNA and uses Arabidopsis e IF2αmolecules as direc substrates[J].Plant Biology,(15):13-18.
Powley I R,Kondrashov A,Young L A,et al.2009.Translational reprogramming following UVB irradiation is mediated by DNA-PKcs and allows selective recruitmen to the polysomes of mRNAs encoding DNA repair enzymes[J].Genes Elopementent,23(10):1207-1220.
Gupta P V,Sahai R,Bhatnagar.2001.Enhanced expression of the recombinant lethal factor of Bacillus anthracis by Fed-Batch culture[J].Biochemical and Biophysical Research Communications,285(4):1025-1033.
Sattlegger E,Hinnebusch A G.2000.Separate domains in GCN1 for binding protein kinase GCN2 and ribosomes are required for GCN2 activation in amino acid-starved cells[J].The EMBO Journal,19(23):6622-6633.
Sonenberg N,Hinnebusch A G.2009.Regulation of translation initiation in eukaryotes:Mechanisms and biological targets[J].Cell,136(4):731-745.
Ventura Salvador.2005.Sequence determinants of protein aggregation:Tools to increase protein solubility[J].Microbial Cell Factories,4(1):1-8.
Wek R C,Jiang H Y,Anthony T G.2006.Coping with stress:eIF2 kinases and translational control[J].Biochemical Society Transactions,34(1):7-11.
Zhang Y H,Wang Y F,Kostya Kanyuka,et al.2009.GCN2-dependent phosphorylation of eukaryotic translation initiation factor-2a in Arabidopsis[J].Journal of Experimental Botany,59(11):3131-3141.
Zhang Y,Dickinson J R,Paul M J,et al.2003.Molecular cloning of an Arabidopsis homologue of GCN2,a protein kinase involved in coordinated response to amino acid starvation[J].Planta,217(4):668-675
Zhang Y,Wang Y,Kanyuka K,et al.2008.GCN2-dependent phosphorylation of eukaryotic translation initiation factor-2αin Arabidopsis[J].Journal of Experimental Botany,59(11):3131-3141.
陈浩,陈于红,朱德煦,等.2002.重组蛋白质纯化技术[J].中国生物工程杂志,22(5):87-92.(Chen H,Chen Y HZhu D X,et al.2002.Recombinant protein purification technology[J].China Bioengineering,22(5):87-92.)
陈明,陈文静.2011.几种微生物表达系统的比较[J].安徽农学通报,17(03):68-71.(Chen M,Chen W J.2011.Comparison of several microbial expression systems[J].Anhui Agricultural Science Bulletin,17(03):68-71.)
韩学波,许崇波,曾瑾.2006.大肠杆菌中外源基因的表达调节[J].生物技术通讯,17(03):418-420.(Han X B,Xu CB,Zeng J.2006.Expression regulation of foreign genes in E.coli[J].Letters in Biotechnology,17(03):418-420.)
何伟.2017.重组大肠杆菌表达包涵体药物蛋白的复性与纯化研究[D].硕士学位论文,华东理工大学,导师:王学东,pp.18-21.(He W.2017.Refolding and purification of recombinant E.coli expressing inclusion body drug proteins[D].Thesis for M.S.,East China University of Science and Technology,Supervisor:Wang X D,pp.18-21.)
黄传臻,刘香利,曹汝菲,等.2017.小麦CWI-B1的原核表达、纯化与多克隆抗体制备[J].农业生物技术学报,25(07):1102-1110.(Huang Z Z,Liu X L,Cao R F,et al.2017.Prokaryotic expression and purification of wheat CWI-B1 and preparation of polyclonal antibodies[J].Journal of Agricultural Biotechnology,25(07):1102-1110.)
卢晟晔,王丽颖.2006.大肠杆菌中外源蛋白高效表达的影响因素及策略研究的新进展[J].中国实验诊断学,10(09):1100-1103.(Lu S Y,Wang L Y.2006.New progress in influencing factors and strategies of overexpression of foreign proteins in E.coli[J].Chinese Journal of Laboratory Diagnosis,10(09):1100-1103.)
李厚华,孔伟胜,魏圆圆,等.2015.植物蛋白质翻译水平的调控[J].植物生理学报,51(2):159-165.(Li H H,Kong W S,Wei Y Y,et al.2015.Regulation of protein translation level in plants[J].Plant Physiology Journal,51(2):159-165.)
许文涛,芦云,罗云波,等.2008.潮霉素磷酸转移酶(hpt)基因的原核表达、蛋白纯化及其抗体制备[J].农业生物技术学报,16(4):647-652.(Xu W T,Lu Y,Luo Y B,et al.2008.Expression of hygromycin-β-phosphotransferase(hpt)gene in Escherichia coli,purification of HPTprotein and preparation of its polyclonal antibodies[J].Journal of Agricultural Biotechnology,16(4):647-652.)
张康旭,刘国顺,李雯雯,等.2014.烟草NtGCN2的克隆与表达分析[J].植物生理学报,50(9):1406-1412.(Zhang K X,Liu G S,Li W W,et al.2014.Cloning and expression analysi s of NtGCN2 in Nicotiana tabacum[J].Plant Physiology Journal,50(9):1406-1412.)
Cherkasova V A,Hinnebusch A G.2003.Translational control by TOR and TAP42 through dephosphorylation of eIF2alpha kinase GCN2[J].Genes Development,17(7):859-872.
Donnelly N,Gorman A M,Gupta S,et al.2013.The e IF2αkinases:Their structures and functions[J].Cellular and Molecular Life Sciences,70(19):3493-3511.
Garcia M A,Meurs E F,Esteban M.2007.The dsRNA protein kinase PKR:Virus and cell control[J].Biochimie,89(6-7):799-811.
Grallert B,Boye E.2013.GCN2,an old dog with new tricks[J].Biochemical Society Transactions,41(6):1687-1691
Halford N G.2006.Regulation of carbon and amino acid metabolism,roles of sucrose nonfermenting-1-related protein kinase-1 and general control nonderepressible-2-related protein kinase[J].Advance in Botanical Research43(05):93-142.
Hey S J,Byrne E,Halford N G.2010.The interface between metabolic and stress signaling[J].Annals of Botany,105(2):197-203.
Hinnebusch A G.2005.Translational regulation of GCN4 and the general amino acid control of yeast[J].Annual Review of Microbiology,59(1):407-450.
Kimball S R.1999.Eukaryotic initiation factor eIF2[J].International Journal of Biochemistry,31(1):25-29.
Lageix S,Rothenburg S,Dever T E,et al.2014.Enhanced interaction between pseudokinase and kinase domains in GCN2 stimulates eIF2αphosphory lation in starved cells[J].PLoS Genetics,10(5):e1004326.
Liu J,Cai C G,Guo Q,et al.2013.Secretory expression and efficient purification of recombinant anthrax toxin letha factor with full biological activity in E.coli.[J].Protein Expression and Purification,89(1):56-61.
Li M W,Auyeung W K,Lam H M.2013.The GCN2 homologue in Arabidopsis thaliana interacts with uncharged tRNA and uses Arabidopsis e IF2αmolecules as direc substrates[J].Plant Biology,(15):13-18.
Powley I R,Kondrashov A,Young L A,et al.2009.Translational reprogramming following UVB irradiation is mediated by DNA-PKcs and allows selective recruitmen to the polysomes of mRNAs encoding DNA repair enzymes[J].Genes Elopementent,23(10):1207-1220.
Gupta P V,Sahai R,Bhatnagar.2001.Enhanced expression of the recombinant lethal factor of Bacillus anthracis by Fed-Batch culture[J].Biochemical and Biophysical Research Communications,285(4):1025-1033.
Sattlegger E,Hinnebusch A G.2000.Separate domains in GCN1 for binding protein kinase GCN2 and ribosomes are required for GCN2 activation in amino acid-starved cells[J].The EMBO Journal,19(23):6622-6633.
Sonenberg N,Hinnebusch A G.2009.Regulation of translation initiation in eukaryotes:Mechanisms and biological targets[J].Cell,136(4):731-745.
Ventura Salvador.2005.Sequence determinants of protein aggregation:Tools to increase protein solubility[J].Microbial Cell Factories,4(1):1-8.
Wek R C,Jiang H Y,Anthony T G.2006.Coping with stress:eIF2 kinases and translational control[J].Biochemical Society Transactions,34(1):7-11.
Zhang Y H,Wang Y F,Kostya Kanyuka,et al.2009.GCN2-dependent phosphorylation of eukaryotic translation initiation factor-2a in Arabidopsis[J].Journal of Experimental Botany,59(11):3131-3141.
Zhang Y,Dickinson J R,Paul M J,et al.2003.Molecular cloning of an Arabidopsis homologue of GCN2,a protein kinase involved in coordinated response to amino acid starvation[J].Planta,217(4):668-675
Zhang Y,Wang Y,Kanyuka K,et al.2008.GCN2-dependent phosphorylation of eukaryotic translation initiation factor-2αin Arabidopsis[J].Journal of Experimental Botany,59(11):3131-3141.