重组人PLCζ蛋白在昆虫细胞/杆状病毒表达系统内的表达、纯化及活性测定
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摘要
PLCζ是PLC家族的一种新型同工酶,在哺乳动物卵母细胞激活中起着极其重要的作用。近年来,体外大量表达和纯化有活性的PLCζ蛋白用于结构生物学研究一直未能获得成功。本研究首次在杆状病毒表达系统中表达和纯化重组人PLCζ蛋白,首先将人PLCζ基因克隆至pFastBac-HTA质粒构建重组载体,转化DH10Bac发生位点特异性转座,经抗性和蓝白斑筛选,获得重组穿梭质粒Bacmid-PLCζ;在脂质体介导下将穿梭质粒转染Sf9昆虫细胞产生重组病毒,扩增病毒感染Sf9昆虫细胞进行蛋白表达;利用Ni~(2+)亲和柱及分子筛来纯化蛋白,并通过考马斯亮蓝染色、Western blotting及飞行时间质谱对蛋白进行鉴定,并进行酶活性测定。结果显示重组蛋白在Sf9昆虫细胞感染杆状病毒后72 h达到峰值并以分泌形式表达在细胞培养基中,Ni~(2+)亲和柱及分子筛纯化后的重组蛋白经Western blotting及电离飞行时间质谱鉴定为PLCζ蛋白,酶活性可达326.8 U/mL。该实验结果为重组人PLCζ蛋白大规模生产和生物医学应用研究提供了可参考利用的技术。
PLCζ is a new isoenzyme of the PLC family which plays an important role in activating mammalian oocytes. In recent years, large-scale expression and purification of active PLCζ protein in vitro for structural biology research has not been successful. In this study, the recombinant human PLCζ protein was expressed and purified in the baculovirus expression system. First, the full length of human PLCζ gene was cloned into the pFastBac-HTA plasmid to form the recombinant donor plasmid that was further transformed into DH10 Bac Escherichia coli cells to construct the recombined bacmid by the site-specific transposition that was screened by resistance and blue-white spots. Then the bacmid was transfected to Sf9 insect cells via cellfectin to package the recombinant baculovirus. After the amplification of the recombinant baculovirous, the recombinant protein was expressed from the cells transduced by the recombinant baculovirus and was purified by Ni-NTA resin. Purified protein was identified by Western blotting and time-of-flight mass spectrometry and the enzyme activity was determined. The results showed that the recombinant PLCζ protein in the Sf9 cells was achieved at 72 hours after baculovirus infection and expressed in secreted form in cell culture medium. The recombinant protein purified by Ni2+ affinity column was identified as PLCζ by Western blotting and ionization time-of-flight mass spectrometry and the enzyme activity was up to326.8 U/mL. The experimental results provide a reference for the large-scale production and biological application of recombinant human PLCζ protein.
PLCζ is a new isoenzyme of the PLC family which plays an important role in activating mammalian oocytes. In recent years, large-scale expression and purification of active PLCζ protein in vitro for structural biology research has not been successful. In this study, the recombinant human PLCζ protein was expressed and purified in the baculovirus expression system. First, the full length of human PLCζ gene was cloned into the pFastBac-HTA plasmid to form the recombinant donor plasmid that was further transformed into DH10 Bac Escherichia coli cells to construct the recombined bacmid by the site-specific transposition that was screened by resistance and blue-white spots. Then the bacmid was transfected to Sf9 insect cells via cellfectin to package the recombinant baculovirus. After the amplification of the recombinant baculovirous, the recombinant protein was expressed from the cells transduced by the recombinant baculovirus and was purified by Ni-NTA resin. Purified protein was identified by Western blotting and time-of-flight mass spectrometry and the enzyme activity was determined. The results showed that the recombinant PLCζ protein in the Sf9 cells was achieved at 72 hours after baculovirus infection and expressed in secreted form in cell culture medium. The recombinant protein purified by Ni2+ affinity column was identified as PLCζ by Western blotting and ionization time-of-flight mass spectrometry and the enzyme activity was up to326.8 U/mL. The experimental results provide a reference for the large-scale production and biological application of recombinant human PLCζ protein.
引文
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[3]Xu HY,Deng K,Luo QB,et al.High Serum FSH is associated with brown oocyte formation and a lower pregnacy rate in human IVF parctice.Cell Physiol Biochem,2016,39(2):677-684.
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[6]Ramadan WM,Kashir J,Jones C,et al.Oocyte activation and phospholipase C zeta(PLCζ):diagnostic and therapeutic implications for assisted reproductive technology.Cell Commun Signal,2012,10(1):12.
[7]Ozil JP,Banrezes B,Tóth S,et al.Ca2+oscillatory pattern in fertilized mouse eggs affects gene expression and development to term.Dev Biol,2006,300(2):534-544.
[8]Tavalaee M,Nasr-Esfahani MH.Expression profile of PLCζ,PAWP,and TR-KIT in association with fertilization potential,embryo development,and pregnancy outcomes in globozoospermic candidates for intra-cytoplasmic sperm injection and artificial oocyte activation.Andrology,2016,4(5):850-856.
[9]Amdani SN,Yeste M,Jones C,et al.Phospholipase C zeta(PLCζ)and male infertility:clinical update and topical developments.Adv Biol Regul,2016,61:58-67.
[10]Yoon SY,Eum JH,Lee JE,et al.Recombinant human phospholipase C zeta 1 induces intracellular calcium oscillations and oocyte activation in mouse and human oocytes.Hum Reprod,2012,27(6):1768-1780.
[11]Nomikos M,Yu YS,Elgmati K,et al.Phospholipase Cζrescues failed oocyte activation in a prototype of male factor infertility.Fertil Steril,2013,99(1):76-85.
[12]Li ZH,Wang QJ.Expression,purification and characterization of catalytic domain of protein kinase D1 in baculovirus-insect cell expression system.Chin J Biotech,2014,30(8):1291-1298(in Chinese).李志红,Wang QJ.蛋白激酶D1催化结构域在昆虫细胞/杆状病毒表达系统内的表达、纯化和活性测定.生物工程学报,2014,30(8):1291-1298.
[13]Luckow VA,Lee SC,Barry GF,et al.Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli.J Virol,1993,67(8):4566-4579.
[14]Baer A,Kehn-Hall K.Viral concentration determination through plaque assays:using traditional and novel overlay systems.J Vis Exp,2014,(93):e52065.
[15]Flieger A,Gong SM,Faigle M,et al.Critical evaluation of p-nitrophenylphosphorylcholine(p-NPPC)as artificial substrate for the detection of phospholipase C.Enzyme Microb Technol,2000,26(5/6):451-458.
[16]Kashir J,Heindryckx B,Jones C,et al.Oocyte activation,phospholipase C zeta and human infertility.Hum Reprod Update,2010,16(6):690-703.
[17]Parrington J.Does a soluble sperm factor trigger calcium release in the egg at fertilization?J Androl,2001,22(1):1-11.
[18]Rogers NT,Halet G,Piao Y,et al.The absence of a Ca2+signal during mouse egg activation can affect parthenogenetic preimplantation development,gene expression patterns,and blastocyst quality.Reproduction,2006,132(1):45-57.
[19]Kouchi Z,Fukami K,Shikano T,et al.Recombinant phospholipase Czeta has high Ca2+sensitivity and induces Ca2+oscillations in mouse eggs.J Biol Chem,2004,279(11):10408-10412.
[20]Lin SY,Chen GY,Hu YC.Recent patents on the baculovirus systems.Recent Pat Biotechnol,2011,5(1):1-11.
[21]Zhang ZG,Jing Y,Barford D.Recombinant expression and reconstitution of multiprotein complexes by the USER cloning method in the insect cell-baculovirus expression system.Methods,2016,95:13-25.
[22]Kool M,Ahrens CH,Vlak JM,et al.Replication of baculovirus DNA.J Gen Virol,1995,76(9):2103-2118.
[23]Ren YY,Chen D,Guo YZ,et al.Expression of human retinol-binding protein 4 in insect baculovirus system and preparation of its polyclonal antibody.Chin J Biotech,2013,29(7):974-985(in Chinese).任玉莹,陈丹,郭玉争,等.人视黄醇结合蛋白4在杆状病毒系统中的表达及其多克隆抗体制备.生物工程学报,2013,29(7):974-985.
[24]Jarvis DL.Developing baculovirus-insect cell expression systems for humanized recombinant glycoprotein production.Virology,2003,310(1):1-7.
[25]Wingfield PT.Overview of the purification of recombinant proteins.Curr Protoc Protein Sci,2015,80(6):1-35.
[2]Swann K,Saunders CM,Rogers NT,et al.PLCζ(zeta):a sperm protein that triggers Ca2+oscillations and egg activation in mammals.Semin Cell Dev Biol,2006,17(2):264-273.
[3]Xu HY,Deng K,Luo QB,et al.High Serum FSH is associated with brown oocyte formation and a lower pregnacy rate in human IVF parctice.Cell Physiol Biochem,2016,39(2):677-684.
[4]Nomikos M,Elgmati K,Theodoridou M,et al.Male infertility-linked point mutation disrupts the Ca2+oscillation-inducing and PIP2,hydrolysis activity of sperm PLCζ.Biochem J,2011,434(2):211-217.
[5]Heytens E,Parrington J,Coward K,et al.Reduced amounts and abnormal forms of phospholipase Czeta(PLCζ)in spermatozoa from infertile men.Hum Reprod,2009,24(10):2417-2428.
[6]Ramadan WM,Kashir J,Jones C,et al.Oocyte activation and phospholipase C zeta(PLCζ):diagnostic and therapeutic implications for assisted reproductive technology.Cell Commun Signal,2012,10(1):12.
[7]Ozil JP,Banrezes B,Tóth S,et al.Ca2+oscillatory pattern in fertilized mouse eggs affects gene expression and development to term.Dev Biol,2006,300(2):534-544.
[8]Tavalaee M,Nasr-Esfahani MH.Expression profile of PLCζ,PAWP,and TR-KIT in association with fertilization potential,embryo development,and pregnancy outcomes in globozoospermic candidates for intra-cytoplasmic sperm injection and artificial oocyte activation.Andrology,2016,4(5):850-856.
[9]Amdani SN,Yeste M,Jones C,et al.Phospholipase C zeta(PLCζ)and male infertility:clinical update and topical developments.Adv Biol Regul,2016,61:58-67.
[10]Yoon SY,Eum JH,Lee JE,et al.Recombinant human phospholipase C zeta 1 induces intracellular calcium oscillations and oocyte activation in mouse and human oocytes.Hum Reprod,2012,27(6):1768-1780.
[11]Nomikos M,Yu YS,Elgmati K,et al.Phospholipase Cζrescues failed oocyte activation in a prototype of male factor infertility.Fertil Steril,2013,99(1):76-85.
[12]Li ZH,Wang QJ.Expression,purification and characterization of catalytic domain of protein kinase D1 in baculovirus-insect cell expression system.Chin J Biotech,2014,30(8):1291-1298(in Chinese).李志红,Wang QJ.蛋白激酶D1催化结构域在昆虫细胞/杆状病毒表达系统内的表达、纯化和活性测定.生物工程学报,2014,30(8):1291-1298.
[13]Luckow VA,Lee SC,Barry GF,et al.Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli.J Virol,1993,67(8):4566-4579.
[14]Baer A,Kehn-Hall K.Viral concentration determination through plaque assays:using traditional and novel overlay systems.J Vis Exp,2014,(93):e52065.
[15]Flieger A,Gong SM,Faigle M,et al.Critical evaluation of p-nitrophenylphosphorylcholine(p-NPPC)as artificial substrate for the detection of phospholipase C.Enzyme Microb Technol,2000,26(5/6):451-458.
[16]Kashir J,Heindryckx B,Jones C,et al.Oocyte activation,phospholipase C zeta and human infertility.Hum Reprod Update,2010,16(6):690-703.
[17]Parrington J.Does a soluble sperm factor trigger calcium release in the egg at fertilization?J Androl,2001,22(1):1-11.
[18]Rogers NT,Halet G,Piao Y,et al.The absence of a Ca2+signal during mouse egg activation can affect parthenogenetic preimplantation development,gene expression patterns,and blastocyst quality.Reproduction,2006,132(1):45-57.
[19]Kouchi Z,Fukami K,Shikano T,et al.Recombinant phospholipase Czeta has high Ca2+sensitivity and induces Ca2+oscillations in mouse eggs.J Biol Chem,2004,279(11):10408-10412.
[20]Lin SY,Chen GY,Hu YC.Recent patents on the baculovirus systems.Recent Pat Biotechnol,2011,5(1):1-11.
[21]Zhang ZG,Jing Y,Barford D.Recombinant expression and reconstitution of multiprotein complexes by the USER cloning method in the insect cell-baculovirus expression system.Methods,2016,95:13-25.
[22]Kool M,Ahrens CH,Vlak JM,et al.Replication of baculovirus DNA.J Gen Virol,1995,76(9):2103-2118.
[23]Ren YY,Chen D,Guo YZ,et al.Expression of human retinol-binding protein 4 in insect baculovirus system and preparation of its polyclonal antibody.Chin J Biotech,2013,29(7):974-985(in Chinese).任玉莹,陈丹,郭玉争,等.人视黄醇结合蛋白4在杆状病毒系统中的表达及其多克隆抗体制备.生物工程学报,2013,29(7):974-985.
[24]Jarvis DL.Developing baculovirus-insect cell expression systems for humanized recombinant glycoprotein production.Virology,2003,310(1):1-7.
[25]Wingfield PT.Overview of the purification of recombinant proteins.Curr Protoc Protein Sci,2015,80(6):1-35.
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