苦参双功能核酸酶1的体外表达及结构性质分析
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摘要
植物双功能核酸酶1影响植株生长、发育及衰老,且研究表明其具有抗肿瘤活性。基于苦参转录组数据克隆了苦参(Sophora flavescens Ait.)中双功能核酸酶1基因的开放读码框(SfBFN1),分析其编码蛋白的理化性质和结构特点。采用RT-PCR方法扩增SfBFN1基因片段,纯化后与pET22b (+)载体连接并转化到大肠杆菌感受态细胞BL21,并以不同的诱导条件,筛选其体外表达的最适宜条件。结果获得了SfBFN1的开放读码框片段,长度为984 bp,编码327个氨基酸,SfBFN1蛋白分子质量约为36.2 ku;利用PHYRE2在线软件预测SfBFN1蛋白的部分三级结构,其富含α螺旋与β折叠,5个α螺旋包裹着7个β折叠,构成了蛋白质立体结构的框架。研究为SfBFN1蛋白开发成为抗癌蛋白质药物制剂奠定了实验基础。
Plant bifunctional nuclease 1 enzymes are responsible for plant growth and developmental processes, including senescence, and they also have anti-tumor effects in vitro. Based on the unigene sequences of the Sophora flavescens transcriptome data, the open reading frame(ORF) of bifunctional nuclease 1 gene(SfBFN1) from Sophora flavescens was cloned, and the physicochemical properties and structural characteristics of the encoded protein were analyzed. SfBFN1 was amplified by RT-PCR and further ligated into pET22 b(+) vector, the recombinant plasmids were then transformed into BL21 competent cells. Optimization of the expression conditions for the SfBFN1 gene was performed at different temperatures and IPTG concentrations. The ORF fragment of SfBFN1 was 984 bp encoding SfBFN1 with 327 amino acids, which molecular weight was about 36.2 ku. SfBFN1 and the bifunctional nuclease 1 from Lupinus angustifolius showed the smallest genetic distance by the evolutionary analysis. The tertiary structure of SfBFN1 was predicted by PHYRE2 online software. The structure is rich in α helix and β fold, with five α helices wrapping seven β folds, forming the frame of the third dimension structure of the protein. The physicochemical properties and structural characteristics of SfBFN1 protein may lay the foundation for further studies on the protein as a potential anti-cancer drug.
Plant bifunctional nuclease 1 enzymes are responsible for plant growth and developmental processes, including senescence, and they also have anti-tumor effects in vitro. Based on the unigene sequences of the Sophora flavescens transcriptome data, the open reading frame(ORF) of bifunctional nuclease 1 gene(SfBFN1) from Sophora flavescens was cloned, and the physicochemical properties and structural characteristics of the encoded protein were analyzed. SfBFN1 was amplified by RT-PCR and further ligated into pET22 b(+) vector, the recombinant plasmids were then transformed into BL21 competent cells. Optimization of the expression conditions for the SfBFN1 gene was performed at different temperatures and IPTG concentrations. The ORF fragment of SfBFN1 was 984 bp encoding SfBFN1 with 327 amino acids, which molecular weight was about 36.2 ku. SfBFN1 and the bifunctional nuclease 1 from Lupinus angustifolius showed the smallest genetic distance by the evolutionary analysis. The tertiary structure of SfBFN1 was predicted by PHYRE2 online software. The structure is rich in α helix and β fold, with five α helices wrapping seven β folds, forming the frame of the third dimension structure of the protein. The physicochemical properties and structural characteristics of SfBFN1 protein may lay the foundation for further studies on the protein as a potential anti-cancer drug.
引文
[1]FARAGE-BARHOM S, BURD S, SONEGO L, et al. Localization of the Arabidopsis, senescence-and cell death-associated BFN1 nuclease: from the ER to fragmented nuclei[J]. Molecular Plant, 2011, 4(6):1062.
[2]SHEN R, LI J, YE D, et al. Combination of onconase and dihydroartemisinin synergistically suppresses growth and angiogenesis of non small-cell lung carcinoma and malignant mesothelioma[J]. Acta Biochim Biophys Sin, 2016, 48(10):894-901.
[3]ZWOLI■SKA M, SMOLEWSKI P. Onconase: a ribonuclease with antitumor activity[J]. Post?py Higieny I Medycyny Doswiadczalnej, 2010, 64(840537):58-66.
[4]FAGAGNINI A, PICA A, FASOLI S, et al. Onconase dimerization through 3D domain swapping: structural investigations and increase of the apoptotic effect in cancer cells[J]. Biochemical Journal, 2017, 474(22): 3767-3781.
[5]VERT A, CASTRO J, RIBó M, et al. Activating transcription factor 3 is crucial for antitumor activity and to strengthen the antiviral properties of Onconase[J]. Oncotarget, 2017, 8(7):11692-11707.
[6]FIORINI C, GOTTE G, DONNARUMMA F, et al. Bovine seminal ribonuclease triggers Beclin1-mediated autophagic cell death in pancreatic cancer cells[J]. Biochimica Et Biophysica Acta, 2014, 1843(5):976.
[7]LIPOVOVA P, PODZIMEK T, ORCTOVA L, et al. Antitumor and biological effects of black pine (Pinus nigra) pollen nuclease[J]. Neoplasma, 2008, 55(2):158-164.
[8]SOUCEK J, SKVOR J, POUCKOVá P, et al. Mung bean sprout (Phaseolus aureus) nuclease and its biological and antitumor effects[J]. Neoplasma, 2006, 53(5):402-409.
[9]MATOUSEK J, PODZIMEK T, POUCKOV P, et al. Antitumor activity of apoptotic nuclease TBN1 from L. esculentum[J]. Neoplasma, 2010, 57(4):339-348.
[10]MATOUSEK J, PODZIMEK T, POUCKOVá P, et al. Antitumor effects and cytotoxicity of recombinant plant nucleases[J]. Oncology Research, 2009, 18(4):163-171.
[11]国家药典委员会. 中华人民共和国药典[M].北京:中国医药科技出版社, 2015:202-203.
[12]JIN J H, KIM J S, KANG S S, et al. Anti-inflammatory and anti-arthritic activity of total flavonoids of the roots of Sophora flavescens[J]. Journal of Ethnopharmacology, 2010, 127(3):589-595.
[13]WANG W, YOU R L, QIN W J, et al. Anti-tumor activities of active ingredients in compound kushen injection[J]. Acta Pharmacologica Sinica, 2015, 36(6):676-679.
[14]LIU D, CHAN B C, CHENG L, et al. Sophora flavescens protects against mycobacterial trehalose dimycolate-induced lung granuloma by inhibiting inflammation and infiltration of macrophages.[J]. Scientific Reports, 2018, 8(1):3903.
[15]ARTIMO P, JONNALAGEDDA M, ARNOLD K, et al. ExPASy: SIB bioinformatics resource portal[J]. Nucleic Acids Research, 2012, 40:597-603.
[16]TAMURA K, PETERSON D, PETERSON N, et al. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[J]. Molecular Biolgy and Evolution, 2011, 28 (10):2731-2739.
[17]CABRERA á C, GIL-REDONDO R, PERONA A, et al. VSDMIP 1.5: an automated structure- and ligand-based virtual screening platform with a PyMOL graphical user interface[J]. Journal of Computer-Aided Molecular Design, 2011, 25(9):813-824.
[18]OLIVEIRA S H, FERRAZ F A, HONORATO R V, et al. KVFinder: steered identification of protein cavities as a PyMOL plugin[J]. BMC Bioinformatics, 2014, 15(1):1-8.
[19]FENDRYCH M, VAN HAUTEGEM T, VAN DURME M, et al. Programmed cell death controlled by ANAC033/SOMBRERO determines root cap organ size in Arabidopsis[J]. Current Biology, 2014, 24(9):931-940.
[20]SAKAMOTO W, TAKAMI T. Nucleases in higher plants and their possible involvement in DNA degradation during leaf senescence[J]. Journal of Experimental Botany, 2014, 65(14): 3835-3843.
[21]MATALLANA-RAMIREZ L P, RAUF M, FARAGE-BARHOM S, et al. NAC transcription factor ORE1 and senescence-induced bifunctional nuclease 1(BFN1) constitute a regulatory cascade in Arabidopsis[J]. Molecular Plant, 2013, 6(5):1438-1452.
[22]FARAGE-BARHOM S, BURD S, SONEGO L, et al. Expression analysis of the BFN1 nuclease gene promoter during senescence, abscission, and programmed cell death-related processes[J]. Journal of Experimental Botany, 2008, 59(12):3247-3258.
[23]LIEBERS N, HOLLAND-LETZ T, WELSCHOF M, et al. Highly efficient destruction of squamous carcinoma cells of the head and neck by photochemical internalization of Ranpirnase[J]. Journal of Expevimegtal Therapeutics & Oncology, 2017, 12(2):113-120.
[24]SQUIQUERA L, TAXMAN D J, BRENDLE S A, et al. Ranpirnase eradicates human papillomavirus in cultured cells and heals anogenital warts in a Phase I study[J]. Antiviral Therapy, 2017, 22(3):247-255.
[25]MATOUSEK J, MATOUSEK J. Plant ribonucleases and nucleases as antiproliferative agens targeting human tumors growing in mice[J]. Recent Pat DNA Gene Seq, 2010, 4(1):29-39.
[26]SICA F, DI-FIORE A, MERLINO A, et al. Structure and stability of the non-covalent swapped dimer of bovine seminal ribonuclease: an enzyme tailored to evade ribonuclease protein inhibitor[J]. Journal of Biological Chemistry, 2004, 279(35):36753-36760.
[27]KOVAL T, LIPOVOVá P, PODZIMEK T, et al. Crystallization of recombinant bifunctional nuclease TBN1 from tomato[J]. Acta Crystallogr Sect F Struct Biol Cryst Commun, 2011, 67(1):124-128.
[28]KOVAL T, LIPOVOVá P, PODZIMEK T, et al. Plant multifunctional nuclease TBN1 with unexpected phospholipase activity: structural study and reaction-mechanism analysis[J]. Acta Crystallogr D Biol Crystallogr, 2013, 69(2):213-226.
[29]PODZIMEK T, MATOU?EK J, LIPOVOVá P, et al. Biochemical properties of three plant nucleases with anticancer potential[J]. Plant Science, 2011, 180(2):343-351.
[30]STRáNSK■ J, KOVAL T, PODZIMEK T, et al. Phosphate binding in the active centre of tomato multifunctional nuclease TBN1 and analysis of superhelix formation by the enzyme[J]. Acta Crystallographica, 2015, 71(11):1408.
[2]SHEN R, LI J, YE D, et al. Combination of onconase and dihydroartemisinin synergistically suppresses growth and angiogenesis of non small-cell lung carcinoma and malignant mesothelioma[J]. Acta Biochim Biophys Sin, 2016, 48(10):894-901.
[3]ZWOLI■SKA M, SMOLEWSKI P. Onconase: a ribonuclease with antitumor activity[J]. Post?py Higieny I Medycyny Doswiadczalnej, 2010, 64(840537):58-66.
[4]FAGAGNINI A, PICA A, FASOLI S, et al. Onconase dimerization through 3D domain swapping: structural investigations and increase of the apoptotic effect in cancer cells[J]. Biochemical Journal, 2017, 474(22): 3767-3781.
[5]VERT A, CASTRO J, RIBó M, et al. Activating transcription factor 3 is crucial for antitumor activity and to strengthen the antiviral properties of Onconase[J]. Oncotarget, 2017, 8(7):11692-11707.
[6]FIORINI C, GOTTE G, DONNARUMMA F, et al. Bovine seminal ribonuclease triggers Beclin1-mediated autophagic cell death in pancreatic cancer cells[J]. Biochimica Et Biophysica Acta, 2014, 1843(5):976.
[7]LIPOVOVA P, PODZIMEK T, ORCTOVA L, et al. Antitumor and biological effects of black pine (Pinus nigra) pollen nuclease[J]. Neoplasma, 2008, 55(2):158-164.
[8]SOUCEK J, SKVOR J, POUCKOVá P, et al. Mung bean sprout (Phaseolus aureus) nuclease and its biological and antitumor effects[J]. Neoplasma, 2006, 53(5):402-409.
[9]MATOUSEK J, PODZIMEK T, POUCKOV P, et al. Antitumor activity of apoptotic nuclease TBN1 from L. esculentum[J]. Neoplasma, 2010, 57(4):339-348.
[10]MATOUSEK J, PODZIMEK T, POUCKOVá P, et al. Antitumor effects and cytotoxicity of recombinant plant nucleases[J]. Oncology Research, 2009, 18(4):163-171.
[11]国家药典委员会. 中华人民共和国药典[M].北京:中国医药科技出版社, 2015:202-203.
[12]JIN J H, KIM J S, KANG S S, et al. Anti-inflammatory and anti-arthritic activity of total flavonoids of the roots of Sophora flavescens[J]. Journal of Ethnopharmacology, 2010, 127(3):589-595.
[13]WANG W, YOU R L, QIN W J, et al. Anti-tumor activities of active ingredients in compound kushen injection[J]. Acta Pharmacologica Sinica, 2015, 36(6):676-679.
[14]LIU D, CHAN B C, CHENG L, et al. Sophora flavescens protects against mycobacterial trehalose dimycolate-induced lung granuloma by inhibiting inflammation and infiltration of macrophages.[J]. Scientific Reports, 2018, 8(1):3903.
[15]ARTIMO P, JONNALAGEDDA M, ARNOLD K, et al. ExPASy: SIB bioinformatics resource portal[J]. Nucleic Acids Research, 2012, 40:597-603.
[16]TAMURA K, PETERSON D, PETERSON N, et al. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[J]. Molecular Biolgy and Evolution, 2011, 28 (10):2731-2739.
[17]CABRERA á C, GIL-REDONDO R, PERONA A, et al. VSDMIP 1.5: an automated structure- and ligand-based virtual screening platform with a PyMOL graphical user interface[J]. Journal of Computer-Aided Molecular Design, 2011, 25(9):813-824.
[18]OLIVEIRA S H, FERRAZ F A, HONORATO R V, et al. KVFinder: steered identification of protein cavities as a PyMOL plugin[J]. BMC Bioinformatics, 2014, 15(1):1-8.
[19]FENDRYCH M, VAN HAUTEGEM T, VAN DURME M, et al. Programmed cell death controlled by ANAC033/SOMBRERO determines root cap organ size in Arabidopsis[J]. Current Biology, 2014, 24(9):931-940.
[20]SAKAMOTO W, TAKAMI T. Nucleases in higher plants and their possible involvement in DNA degradation during leaf senescence[J]. Journal of Experimental Botany, 2014, 65(14): 3835-3843.
[21]MATALLANA-RAMIREZ L P, RAUF M, FARAGE-BARHOM S, et al. NAC transcription factor ORE1 and senescence-induced bifunctional nuclease 1(BFN1) constitute a regulatory cascade in Arabidopsis[J]. Molecular Plant, 2013, 6(5):1438-1452.
[22]FARAGE-BARHOM S, BURD S, SONEGO L, et al. Expression analysis of the BFN1 nuclease gene promoter during senescence, abscission, and programmed cell death-related processes[J]. Journal of Experimental Botany, 2008, 59(12):3247-3258.
[23]LIEBERS N, HOLLAND-LETZ T, WELSCHOF M, et al. Highly efficient destruction of squamous carcinoma cells of the head and neck by photochemical internalization of Ranpirnase[J]. Journal of Expevimegtal Therapeutics & Oncology, 2017, 12(2):113-120.
[24]SQUIQUERA L, TAXMAN D J, BRENDLE S A, et al. Ranpirnase eradicates human papillomavirus in cultured cells and heals anogenital warts in a Phase I study[J]. Antiviral Therapy, 2017, 22(3):247-255.
[25]MATOUSEK J, MATOUSEK J. Plant ribonucleases and nucleases as antiproliferative agens targeting human tumors growing in mice[J]. Recent Pat DNA Gene Seq, 2010, 4(1):29-39.
[26]SICA F, DI-FIORE A, MERLINO A, et al. Structure and stability of the non-covalent swapped dimer of bovine seminal ribonuclease: an enzyme tailored to evade ribonuclease protein inhibitor[J]. Journal of Biological Chemistry, 2004, 279(35):36753-36760.
[27]KOVAL T, LIPOVOVá P, PODZIMEK T, et al. Crystallization of recombinant bifunctional nuclease TBN1 from tomato[J]. Acta Crystallogr Sect F Struct Biol Cryst Commun, 2011, 67(1):124-128.
[28]KOVAL T, LIPOVOVá P, PODZIMEK T, et al. Plant multifunctional nuclease TBN1 with unexpected phospholipase activity: structural study and reaction-mechanism analysis[J]. Acta Crystallogr D Biol Crystallogr, 2013, 69(2):213-226.
[29]PODZIMEK T, MATOU?EK J, LIPOVOVá P, et al. Biochemical properties of three plant nucleases with anticancer potential[J]. Plant Science, 2011, 180(2):343-351.
[30]STRáNSK■ J, KOVAL T, PODZIMEK T, et al. Phosphate binding in the active centre of tomato multifunctional nuclease TBN1 and analysis of superhelix formation by the enzyme[J]. Acta Crystallographica, 2015, 71(11):1408.