麻鸭磷脂氢谷胱甘肽过氧化物酶高效可溶性表达及其部分酶学性质
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摘要
【目的】克隆麻鸭磷脂氢谷胱甘肽过氧化物酶基因(GPx4)并在大肠杆菌中表达,分析其酶学特性,为研究GPx4功能及其在羟基脂肪酸形成过程中的调节机制打下基础。【方法】采用RT-PCR克隆麻鸭GPx4(DGPx4)的编码基因,利用定点突变的方法构建半胱氨酸突变体DGPx4表达载体pMBP-DGPx4(48Sec→Cys),并在大肠杆菌中通过添加His标签进行可溶性表达,经Ni-NTA亲和层析和离子交换层析纯化得到融合蛋白DGPx4;采用总谷胱甘肽过氧化物酶活性检测试剂盒检测DGPx4活力以研究其酶学性质。【结果】克隆获得的DGPx4基因编码序列全长516 bp,编码172个氨基酸,编码蛋白分子量约20 k D,理论等电点(p I)为8.9。构建的突变体基因原核表达载体pMBP-DGPx4(48Sec→Cys)可在大肠杆菌中成功诱导表达获得融合蛋白DGPx4,经Ni-NTA亲和层析和离子交换层析即获得高纯度的DGPx4。以烟酰胺腺嘌呤二核苷酸磷酸(NADPH)为底物时,DGPx4的最适反应温度为32℃,最适pH为8.0,对NaCl浓度较敏感;Cu2+和Ni2+对DGPx4活力有较高的促进作用,Mg2+和Mn2+对DGPx4活力有一定的抑制作用,Ca2+和Zn2+对DGPx4活力则无明显的促进或抑制作用。【结论】从鸭肝细胞中克隆获得的DGPx4基因序列经定点突变后可在原核细胞中高效表达,且纯化后的高纯度融合蛋白DGPx4具有GPx4活力,能在抗脂质氧化过程中发挥作用。
【Objective】The shelduck phospholipid hydroperoxide glutathione peroxidase gene(GPx4)was cloned and expressed in Escherichia coli,and its enzymatic properties were analyzed,which laid a foundation for preliminary study of GPx4 function and its regulation mechanism in the formation of hydroxy fatty acids.【Method】Shelduck GPx4(DGPx4)encoded gene was cloned by RT-PCR,and site-directed mutation was used to established cysteine mutant DGPx4 expression vector pMBP-DGPx4(48 Sec→Cys). His tag was added into E. coli to achieve soluble expression. Fusion protein DGPx4 was obtained by Ni-NTA affinity chromatography and ion exchange chromatography purification. DGPx4 activity and the enzymatic properties were measured by total glutathione peroxidase activity assay kit.【Result】The results showed that the obtained DGPx4 gene coding sequence was 516 bp in length and encoded 172 amino acids. The encoded protein had a molecular weight of approximately 20 kD and a theoretical isoelectric point(pI)of 8.9. The constructed mutant gene prokaryotic expression vector p MBP-DGPx4(48 Sec→Cys)was successfully induced to express the fusion protein DGPx4 in Escherichia coli,and high purity DGPx4 was obtained by Ni-NTA affinity chromatography and ion exchange chromatography. When nicotinamide adenine dinucleotide phosphate(NADPH)was used as the substrate,the optimum reaction temperature for DGPx4 was 32 ℃,and the optimum pH was 8.0,and sensitive to NaCl concentration.Cu2+and Ni2+could activate the activity of DGPx4,Mg2+and Mn2+inhibited its activity,while Ca2+and Zn2+had no obvious effect on DGPx4 activity.【Conclusion】The DGPx4 gene sequence cloned from duck liver cells can be highly expressed in prokaryotic cells after site-directed mutagenesis,and the purified high-purity fusion protein DGPx4 has GPx4 activity and can play a role in anti-lipid oxidation.
【Objective】The shelduck phospholipid hydroperoxide glutathione peroxidase gene(GPx4)was cloned and expressed in Escherichia coli,and its enzymatic properties were analyzed,which laid a foundation for preliminary study of GPx4 function and its regulation mechanism in the formation of hydroxy fatty acids.【Method】Shelduck GPx4(DGPx4)encoded gene was cloned by RT-PCR,and site-directed mutation was used to established cysteine mutant DGPx4 expression vector pMBP-DGPx4(48 Sec→Cys). His tag was added into E. coli to achieve soluble expression. Fusion protein DGPx4 was obtained by Ni-NTA affinity chromatography and ion exchange chromatography purification. DGPx4 activity and the enzymatic properties were measured by total glutathione peroxidase activity assay kit.【Result】The results showed that the obtained DGPx4 gene coding sequence was 516 bp in length and encoded 172 amino acids. The encoded protein had a molecular weight of approximately 20 kD and a theoretical isoelectric point(pI)of 8.9. The constructed mutant gene prokaryotic expression vector p MBP-DGPx4(48 Sec→Cys)was successfully induced to express the fusion protein DGPx4 in Escherichia coli,and high purity DGPx4 was obtained by Ni-NTA affinity chromatography and ion exchange chromatography. When nicotinamide adenine dinucleotide phosphate(NADPH)was used as the substrate,the optimum reaction temperature for DGPx4 was 32 ℃,and the optimum pH was 8.0,and sensitive to NaCl concentration.Cu2+and Ni2+could activate the activity of DGPx4,Mg2+and Mn2+inhibited its activity,while Ca2+and Zn2+had no obvious effect on DGPx4 activity.【Conclusion】The DGPx4 gene sequence cloned from duck liver cells can be highly expressed in prokaryotic cells after site-directed mutagenesis,and the purified high-purity fusion protein DGPx4 has GPx4 activity and can play a role in anti-lipid oxidation.
引文
蔡丝丝,刘金,王林川,陈芳艳.2014.磷脂氢谷胱甘肽过氧化物酶在精子发育中的作用研究进展[J].广东农业科学,41(16):116-119.[Cai S S,Liu J,Wang L C,Chen F Y.2014.Review on the function of phospholipid hydroperoxide glutathione peroxidase in sperm development[J].Guangdong Agricultural Sciences,41(16):116-119.]
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李甜,刘冠兰,段明星,余世实,刘进元,严泽民,徐之伟.2010.原核表达的萝卜PHGPx对黑色素瘤B16细胞紫外辐射损伤的恢复作用[J].中国生物工程杂志,30(2):66-70.[Li T,Liu G L,Duan M X,Yu S S,Liu J Y,Yan ZM,Xu Z W.2010.Effects of prokaryotic expressed radish phospholipid hydroperoxide glutathione peroxidase on restoration of B16 melanoma cells irradiated with ultroviolet[J].China Biotechnology,30(2):66-70.]
李洋,李晖,祝建波,刘进元.2010.重组萝卜磷脂氢谷胱甘肽过氧化物酶在毕赤酵母优化表达初步纯化与鉴定[J].中国生物工程杂志,30(4):54-59.[Li Y,Li H,Zhu J B,Liu J Y.2010.Optimized expression and identification of the radish phospholipid hydrogen glutathione peroxidase in pichia pastoris[J].China Biotechnology,30(4):54-59.]
刘春云,傅明骏,张子平,邹志华,贾锡伟,王艺磊.2015.拟穴青蟹PHGPx基因的克隆及其表达分析[J].水产学报,39(2):161-173.[Liu C Y,Fu M J,Zhang Z P,Zou Z H,Jia X W,Wang Y L.2015.Molecular cloning and expression analysis of Sp-PHGPx in Scylla paramamosain[J].Journal of Fisheries of China,39(2):161-173.]
刘冠兰.2010.萝卜磷脂氢谷胱甘肽过氧化物酶的抗氧化功能研究[D].北京:清华大学.[Liu G L.2010.Study about the antioxidant function of radish phospholipid hydroperoxide glutathione peroxidase[D].Beijing:Tsinghua University.]
施力光,荀文娟,周汉林,侯冠彧,岳文斌,张春香,杨茹洁.2012.山羊磷脂氢谷胱甘肽过氧化物酶生物信息学分析[J].生物技术通报,(12):106-113.[Shi L G,Xun W J,Zhou H L,Hou G X,Yue W B,Zhang C X,Yang R J.2012.Bioinformatis analysis of PHGPx gene in goat[J].Biotechnology Bulletin,(12):106-113.]
闫春燕,徐承水,张士璀.2008.动物磷脂氢谷胱甘肽过氧化物酶研究进展[J].科技信息(科学教研),(22):366.[Yan C Y,Xu C S,Zhang S C.2008.Research progress on animal phospholipid hydrogen glutathione peroxidase[J].Science and Technology Information(Science Teaching and Research),(22):366.]
杨杰,潘孝青,赵艳红,宋丹丹,秦枫,邵乐,李健,张霞,张琳琳,王菁,潘翠玲.2017.硒对新西兰兔皮肤抗氧化性及GPx4、EGFR蛋白表达的影响[J].江苏农业学报,33(1):133-140.[Yang J,Pan X Q,Zhao Y H,Song D D,Qin F,Shao L,Li J,Zhang X,Zhang L L,Wang J,Pan C L.2017.Effects of selenium on skin antioxidant and expression of GPx4 and EGFR proteins in New Zealand rabbit[J].Jiangsu Journal of Agricultural Sciences,33(1):133-140.]
张超,白璐,阳刚,宋旭,殷中琼,邹元峰,魏琴,杜永华.2017.柠檬酵素对麻鸭免疫器官指数和抗氧化功能的影响[J].河南农业大学学报,51(5):667-671.[Zhang C,Bai L,Yang G,Song X,Yin Z Q,Zou Y F,Wei Q,Du Y H.2017.Effects of lemon enzyme on immune organ index and antioxidant capacity of hemp ducks[J].Journal of Henan Agricultural University,51(5):667-671.]
张丹丹,娄鹏博,李振.2012.GPxs家族的研究进展[J].农业技术与装备,(15):66-67.[Zhang D D,Lou P B,Li Z.2012.The progress of GPxs[J].Agricultural Technology and Equipment,(15):66-67.]
张玉梅,李鹏鹏,张牧焓,王晶晶,王道营,徐为民.2018.麻鸭HSP90α基因的原核表达、纯化及磷脂结合活性的鉴定[J].食品科学,39(2):99-104.[Zhang Y M,Li P P,Zhang M H,Wang J J,Wang D Y,Xu W M.2018.Prokaryotic expression,purification and identification of phospholipid binding activity of Anas platyrhynchas HSP90α[J].Food Science,39(2):99-104.]
赵文然,周令望,刘艺,钟学宽.2004.磷脂氢谷胱甘肽过氧化物酶的克隆及在大肠埃希菌中的表达[J].中华地方病学杂志,23(2):119-120.[Zhao W R,Zhou L W,Liu Y,Zhong X K.2004.Cloning and expression of human phospholipid hydroperoxide glutathione peroxidase in Escherichia coli[J].Chinese Jouranl of Endemiology,23(2):119-120.]
Choque B,Catheline D,Rioux V,Legrand P.2014.Linoleic acid:Between doubts and certainties[J].Biochimie,96:14-21.
Imai H,Nakagawa Y.2003.Biological significance of phospholipid hydroperoxide glutathione peroxidase(PHGPx,GPx4)in mammalian cells[J].Free Radical Biology&Medicine,34(2):145-169.
Jira W,Spiteller G,Carson W,Schramm A.1998.Strong increase in hydroxy fatty acids derived from linoleic acid in human low density lipoproteins of atherosclerotic patients[J].Chemistry and Physics of Lipids,91(1):1-11.
Jostarndt K,Gellert N,Rubic T,Weber C,Kühn H,Johansen B,Hrboticky N,Neuzil J.2002.Dissociation of apoptosis induction and CD36 upregulation by enzymatically modified low-density lipoprotein in monocytic cells[J].Biochemical and Biophysical Research Communications,290(3):988-993.
Peng D,Belkhiri A,Hu T,Chaturvedi R,Asim M,Wilson KT,Zaika A,El-Rifai W.2012.Glutathione peroxidase 7protects against oxidative DNA damage in oesophageal cells[J].Gut,61(9):1250-1260.
Scheerer P,Borchert A,Krauss N,Wessner H,Gerth C,H?hne W,Kuhn H.2007.Structural basis for catalytic activity and enzyme polymerization of phospholipid hydroperoxide glutathione peroxidase-4(GPx4)[J].Biochemistry,46(31):9041-9049.
Song H,Wu H H,Geng Z M,Sun C,Ren S,Wang D Y,Zhang M H,Liu F,Xu W M.2016.Simultaneous determination of 13-HODE,9,10-DHODE,and 9,10,13-THODEin cured meat products by LC-MS/MS[J].Food Science,9(10):2832-2841.
Vangaveti V N,Shashidhar V M,Rush C,Malabu U H,Rasalam R R,Collier F,Baune B T,Kennedy R L.2014.Hydroxyoctadecadienoic acids regulate apoptosis in human THP-1 cells in a PPARγ-dependent manner[J].Lipids,49(12):1181-1192.
Wang L,Harris S M,Espinoza H M,Mcclain V,Gallagher EP.2012.Characterization of phospholipid hydroperoxide glutathione metabolizing peroxidase(GPx4)isoforms in Coho salmon olfactory and liver tissues and their modulation by cadmium[J].Aquatic Toxicology,114-115:134-141.doi:10.1016/j.aquatox.2012.02.025.
Yant L J,Ran Q,Rao L,van Remmen H,Shibatani T,Belter JG,Motta L,Richardson A,Prolla T A.2003.The selenoprotein GPx4 is essential for mouse development and protects from radiation and oxidative damage insults[J].Free Radical Biology&Medicine,34(4):496-502.
冯凯,李胜,麻锐,姜锐,孙立伟,陈伟.2016.山参磷脂氢谷胱甘肽过氧化物酶c DNA克隆及序列分析[J].食品工业科技,37(11):152-155.[Feng K,Li S,Ma R,Jiang R,Sun LW,Chen W.2016.Cloning and sequence analysis of wild ginseng phospholipid hydroperoxide glutathione peroxidase cDNA[J].Science and Techonology of Food Industry,37(11):152-155.]
李甜,刘冠兰,段明星,余世实,刘进元,严泽民,徐之伟.2010.原核表达的萝卜PHGPx对黑色素瘤B16细胞紫外辐射损伤的恢复作用[J].中国生物工程杂志,30(2):66-70.[Li T,Liu G L,Duan M X,Yu S S,Liu J Y,Yan ZM,Xu Z W.2010.Effects of prokaryotic expressed radish phospholipid hydroperoxide glutathione peroxidase on restoration of B16 melanoma cells irradiated with ultroviolet[J].China Biotechnology,30(2):66-70.]
李洋,李晖,祝建波,刘进元.2010.重组萝卜磷脂氢谷胱甘肽过氧化物酶在毕赤酵母优化表达初步纯化与鉴定[J].中国生物工程杂志,30(4):54-59.[Li Y,Li H,Zhu J B,Liu J Y.2010.Optimized expression and identification of the radish phospholipid hydrogen glutathione peroxidase in pichia pastoris[J].China Biotechnology,30(4):54-59.]
刘春云,傅明骏,张子平,邹志华,贾锡伟,王艺磊.2015.拟穴青蟹PHGPx基因的克隆及其表达分析[J].水产学报,39(2):161-173.[Liu C Y,Fu M J,Zhang Z P,Zou Z H,Jia X W,Wang Y L.2015.Molecular cloning and expression analysis of Sp-PHGPx in Scylla paramamosain[J].Journal of Fisheries of China,39(2):161-173.]
刘冠兰.2010.萝卜磷脂氢谷胱甘肽过氧化物酶的抗氧化功能研究[D].北京:清华大学.[Liu G L.2010.Study about the antioxidant function of radish phospholipid hydroperoxide glutathione peroxidase[D].Beijing:Tsinghua University.]
施力光,荀文娟,周汉林,侯冠彧,岳文斌,张春香,杨茹洁.2012.山羊磷脂氢谷胱甘肽过氧化物酶生物信息学分析[J].生物技术通报,(12):106-113.[Shi L G,Xun W J,Zhou H L,Hou G X,Yue W B,Zhang C X,Yang R J.2012.Bioinformatis analysis of PHGPx gene in goat[J].Biotechnology Bulletin,(12):106-113.]
闫春燕,徐承水,张士璀.2008.动物磷脂氢谷胱甘肽过氧化物酶研究进展[J].科技信息(科学教研),(22):366.[Yan C Y,Xu C S,Zhang S C.2008.Research progress on animal phospholipid hydrogen glutathione peroxidase[J].Science and Technology Information(Science Teaching and Research),(22):366.]
杨杰,潘孝青,赵艳红,宋丹丹,秦枫,邵乐,李健,张霞,张琳琳,王菁,潘翠玲.2017.硒对新西兰兔皮肤抗氧化性及GPx4、EGFR蛋白表达的影响[J].江苏农业学报,33(1):133-140.[Yang J,Pan X Q,Zhao Y H,Song D D,Qin F,Shao L,Li J,Zhang X,Zhang L L,Wang J,Pan C L.2017.Effects of selenium on skin antioxidant and expression of GPx4 and EGFR proteins in New Zealand rabbit[J].Jiangsu Journal of Agricultural Sciences,33(1):133-140.]
张超,白璐,阳刚,宋旭,殷中琼,邹元峰,魏琴,杜永华.2017.柠檬酵素对麻鸭免疫器官指数和抗氧化功能的影响[J].河南农业大学学报,51(5):667-671.[Zhang C,Bai L,Yang G,Song X,Yin Z Q,Zou Y F,Wei Q,Du Y H.2017.Effects of lemon enzyme on immune organ index and antioxidant capacity of hemp ducks[J].Journal of Henan Agricultural University,51(5):667-671.]
张丹丹,娄鹏博,李振.2012.GPxs家族的研究进展[J].农业技术与装备,(15):66-67.[Zhang D D,Lou P B,Li Z.2012.The progress of GPxs[J].Agricultural Technology and Equipment,(15):66-67.]
张玉梅,李鹏鹏,张牧焓,王晶晶,王道营,徐为民.2018.麻鸭HSP90α基因的原核表达、纯化及磷脂结合活性的鉴定[J].食品科学,39(2):99-104.[Zhang Y M,Li P P,Zhang M H,Wang J J,Wang D Y,Xu W M.2018.Prokaryotic expression,purification and identification of phospholipid binding activity of Anas platyrhynchas HSP90α[J].Food Science,39(2):99-104.]
赵文然,周令望,刘艺,钟学宽.2004.磷脂氢谷胱甘肽过氧化物酶的克隆及在大肠埃希菌中的表达[J].中华地方病学杂志,23(2):119-120.[Zhao W R,Zhou L W,Liu Y,Zhong X K.2004.Cloning and expression of human phospholipid hydroperoxide glutathione peroxidase in Escherichia coli[J].Chinese Jouranl of Endemiology,23(2):119-120.]
Choque B,Catheline D,Rioux V,Legrand P.2014.Linoleic acid:Between doubts and certainties[J].Biochimie,96:14-21.
Imai H,Nakagawa Y.2003.Biological significance of phospholipid hydroperoxide glutathione peroxidase(PHGPx,GPx4)in mammalian cells[J].Free Radical Biology&Medicine,34(2):145-169.
Jira W,Spiteller G,Carson W,Schramm A.1998.Strong increase in hydroxy fatty acids derived from linoleic acid in human low density lipoproteins of atherosclerotic patients[J].Chemistry and Physics of Lipids,91(1):1-11.
Jostarndt K,Gellert N,Rubic T,Weber C,Kühn H,Johansen B,Hrboticky N,Neuzil J.2002.Dissociation of apoptosis induction and CD36 upregulation by enzymatically modified low-density lipoprotein in monocytic cells[J].Biochemical and Biophysical Research Communications,290(3):988-993.
Peng D,Belkhiri A,Hu T,Chaturvedi R,Asim M,Wilson KT,Zaika A,El-Rifai W.2012.Glutathione peroxidase 7protects against oxidative DNA damage in oesophageal cells[J].Gut,61(9):1250-1260.
Scheerer P,Borchert A,Krauss N,Wessner H,Gerth C,H?hne W,Kuhn H.2007.Structural basis for catalytic activity and enzyme polymerization of phospholipid hydroperoxide glutathione peroxidase-4(GPx4)[J].Biochemistry,46(31):9041-9049.
Song H,Wu H H,Geng Z M,Sun C,Ren S,Wang D Y,Zhang M H,Liu F,Xu W M.2016.Simultaneous determination of 13-HODE,9,10-DHODE,and 9,10,13-THODEin cured meat products by LC-MS/MS[J].Food Science,9(10):2832-2841.
Vangaveti V N,Shashidhar V M,Rush C,Malabu U H,Rasalam R R,Collier F,Baune B T,Kennedy R L.2014.Hydroxyoctadecadienoic acids regulate apoptosis in human THP-1 cells in a PPARγ-dependent manner[J].Lipids,49(12):1181-1192.
Wang L,Harris S M,Espinoza H M,Mcclain V,Gallagher EP.2012.Characterization of phospholipid hydroperoxide glutathione metabolizing peroxidase(GPx4)isoforms in Coho salmon olfactory and liver tissues and their modulation by cadmium[J].Aquatic Toxicology,114-115:134-141.doi:10.1016/j.aquatox.2012.02.025.
Yant L J,Ran Q,Rao L,van Remmen H,Shibatani T,Belter JG,Motta L,Richardson A,Prolla T A.2003.The selenoprotein GPx4 is essential for mouse development and protects from radiation and oxidative damage insults[J].Free Radical Biology&Medicine,34(4):496-502.
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