融合蛋白NusA-hRI的高效异源表达、纯化及活性分析
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摘要
人核糖核酸酶抑制因子(human ribonuclease inhibitor,hRI)是一种能够调节核糖核酸酶活性的酸性包浆蛋白。通过构建含SUMO、IF2、GST、NusA、MsyB、Trx和MBP融合标签的重组表达载体,以大肠杆菌BL21(DE3)作为宿主菌进行自诱导(auto-induction,AI)表达,从而使hRI的表达量得以提升。利用MagNi磁珠纯化及电泳分析hRI的表达状况,通过RNase/Sepharose亲和层析获得纯度较高的蛋白。纯化后获得的融合蛋白浓度为2 960. 513mg/L,与其它公司的hRI活性进行比较,检测其酶活性约为50U/μl,并使其成功用于RNA的保护,为Nus A-hRI的应用提供理论依据。
Human ribonuclease inhibitor(hRI) is an acidic pulping protein that regulates ribonuclease activity. By constructing a recombinant expression vector containing SUMO,IF2,GST,Nus A,MsyB,Trx and MBP fusion tags,E. coli BL21(DE3) was used as a host strain for auto-induction(AI) expression,thereby the expression level of hRI is improved. The expression of hRI was analyzed by MagNi magnetic bead purification and electrophoresis,and the protein with higher purity was obtained by RNase/Sepharose affinity chromatography. The concentration of the fusion protein obtained after purification was 2 960. 513 mg/L,which was compared with other companies,and its enzyme activity was about 50 U/μl,which was successfully used for RNA protection.Purely provide a theoretic-al basis for the application of NusA-hRI.
Human ribonuclease inhibitor(hRI) is an acidic pulping protein that regulates ribonuclease activity. By constructing a recombinant expression vector containing SUMO,IF2,GST,Nus A,MsyB,Trx and MBP fusion tags,E. coli BL21(DE3) was used as a host strain for auto-induction(AI) expression,thereby the expression level of hRI is improved. The expression of hRI was analyzed by MagNi magnetic bead purification and electrophoresis,and the protein with higher purity was obtained by RNase/Sepharose affinity chromatography. The concentration of the fusion protein obtained after purification was 2 960. 513 mg/L,which was compared with other companies,and its enzyme activity was about 50 U/μl,which was successfully used for RNA protection.Purely provide a theoretic-al basis for the application of NusA-hRI.
引文
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[3]付大伟,陈启蒙,徐伟.融合蛋白Trx-T4 DL可溶性表达、纯化与其生物活性应用.食品工业科技,2018,39(07):83-89,96.Fu D W,Chen Q M,Xu W.Soluble expression,purification and biological application of fusion protein Trx-T4 DL.Science and Technology of Food Industry,2018,39(07):83-89,96.
[4]Nie Y,Yan W,Xu Y,et al.High-level expression of Bacillus naganoensis pullulanase from recombinant Escherichia coli with auto-induction:Effect of lac operator.Plo S One,2013,8(10):78-84.
[5]Dammicco S,Goux M,Lemair C,et al.Regiospecific radiolabelling of Nanofitin on Ni magnetic beads with[18F]FBEM and in vivo PET studies.Nuclear Medicine&Biology,2017,51(6):33-39.
[6]Makino T,Skretas G,Georgiou G.Strain engineering for improved expression of recombinant proteins in bacteria.Microb Cell Fact,2011,5(7):10-32.
[7]Park J H,Hwang I S,Kim K I,et al.Functional expression of recombinant human ribonuclease/angiogenin inhibitor in stably transformed Drosophila melanogaster S2 cells.Cytotechnology,2008,57(1):93-99.
[8]Klink T A,Vicentini A M,Hofsteenge J,et al.High-level soluble production and characterization of porcine ribonuclease inhibitor.Protein Expr Purif,2001,22(2):174-179.
[9]万鹏.猪流行性腹泻病毒抗原基因COE的原核表达与纯化.郑州:河南农业大学,2015.Wan P.Prokaryotic expression and purification of porcine epidemic diarrhea virus antigen gene COE.Zhengzhou:Henan Agricultural University,2015.
[10]崔倩倩.防治小菜蛾高效绿僵菌工程菌的构建.北京:中国农业科学院,2013.Cui Q Q.Construction of an engineered fungus for the control of Plutella xylostella.Beijing:Chinese Academy of Agricultural Sciences,2013.
[11]顾娟,劳勋,金明飞,等.人胰高血糖素样肽-1突变体融合蛋白在大肠杆菌中的自诱导表达优化.微生物学通报,2010,37(05):726-731.Gu J,Lao X,Jin M F,et al.Optimization of auto-induction expression of human glucagon-like peptide-1 mutant fusion protein in Escherichia coli.Bulletin of Microbiology,2010,37(05):726-731.
[12]付大伟,陈启蒙,孙莹莹,等.T4 DNA聚合酶在大肠杆菌中高效表达、纯化及部分生物学活性研究.食品科学,2018,39(16):214-220.Fu D W,Chen Q M,Sun Y Y,et al.High expression,purification and partial biological activity of T4 DNA polymerase in E.coli.Food Science,2018,39(16):214-220.
[13]Bernier S C,Cantin L,Salesse C.Systematic analysis of the expression,solubility and purification of a passenger protein in fusion with different tags.Protein Expression and Purification,2018,152(7):92-106.
[14]Blackburn P,Wilson G,Moore S.Ribonuclease inhibitor from human placenta.Purification and properties.J Biol Chem,1977,252(16):5904-5910.
[15]Guo W H,Cao L,Jia Z J.High level soluble production of functional ribonuclease inhibitor in Escherichia coli by fusing it to soluble partners.Protein Expression and Purification,2011,77(2):185-192.
[16]刘皓熙.TAT-MT融合蛋白在大肠杆菌中的高效表达、纯化及体外活性初步测定.广州:暨南大学,2011.Liu H X.High-level expression,purification and in vitro activity determination of TAT-MT fusion protein in Escherichia coli.Guangzhou:Jinan University,2011.