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  • CN 31-1691/TQ

重组大肠杆菌表达豹蛙酶发酵培养基和发酵条件的优化

夏祯 赵兴聪 王学东

夏祯, 赵兴聪, 王学东. 重组大肠杆菌表达豹蛙酶发酵培养基和发酵条件的优化[J]. 华东理工大学学报(自然科学版), 2021, 47(5): 592-597. doi: 10.14135/j.cnki.1006-3080.20200824001
引用本文: 夏祯, 赵兴聪, 王学东. 重组大肠杆菌表达豹蛙酶发酵培养基和发酵条件的优化[J]. 华东理工大学学报(自然科学版), 2021, 47(5): 592-597. doi: 10.14135/j.cnki.1006-3080.20200824001
XIA Zhen, ZHAO Xingcong, WANG Xuedong. Optimization of Fermentation Medium and Conditions for Recombinant E. coli Expressing Onconase[J]. Journal of East China University of Science and Technology, 2021, 47(5): 592-597. doi: 10.14135/j.cnki.1006-3080.20200824001
Citation: XIA Zhen, ZHAO Xingcong, WANG Xuedong. Optimization of Fermentation Medium and Conditions for Recombinant E. coli Expressing Onconase[J]. Journal of East China University of Science and Technology, 2021, 47(5): 592-597. doi: 10.14135/j.cnki.1006-3080.20200824001

重组大肠杆菌表达豹蛙酶发酵培养基和发酵条件的优化

doi: 10.14135/j.cnki.1006-3080.20200824001
基金项目: 国家科技重大专项“生物技术药物中试放大及分离纯化技术平台”(2012ZX09304009)
详细信息
    作者简介:

    夏祯:夏 祯(1987-),女,辽宁抚顺人,硕士生,研究方向为重组蛋白药物

    通讯作者:

    王学东,E-mail:xdwang@ecust.edu.cn

  • 中图分类号: Q815

Optimization of Fermentation Medium and Conditions for Recombinant E. coli Expressing Onconase

  • 摘要: 采用Plackett-Burman(PB)实验设计,考察了培养基各组分对重组大肠杆菌表达豹蛙酶的影响,优化了重组大肠杆菌表达豹蛙酶的发酵培养基,并进一步优化了诱导表达条件。在此基础上,采用指数补料和pH-Stat方式在7 L发酵罐中进一步验证优化结果。结果表明:摇瓶水平豹蛙酶表达量由优化前的9%提高到优化后的36%,OD600值由优化前的4.80提升到优化后的5.47。优化后7 L发酵罐中重组菌豹蛙酶表达水平可达到55%,OD600可达到35。

     

  • 图  1  诱导温度对重组菌生长以及表达豹蛙酶的影响

    Figure  1.  Effect of induction temperature on the growth and expression of ranpirnase of recombinant E. coli

    图  2  诱导浓度对重组菌生长以及表达豹蛙酶的影响

    Figure  2.  Effect of induction concentration on the growth and expression of ranpirnase of recombinant E. coli

    图  3  诱导前培养时间对重组菌生长以及表达豹蛙酶的影响

    Figure  3.  Effect of culture time before induction on the growth and expression of ranpirnase of recombinant E. coli

    图  4  摇瓶水平上优化前后重组菌表达豹蛙酶情况

    Figure  4.  Expression of ranpirnase in recombinant E. coli before and after optimization at shake flask level

    1—Fermentation before optimization; 2—Fermentation after optimization; 3—Marker

    图  5  发酵罐水平优化后重组菌表达豹蛙酶情况

    Figure  5.  Expression of ranpirnase by recombinant E.coli after optimization of fermentor

    1—Marker; 2—Recombinant E.coli cell; 3—Supernatant after cell fragmentation; 4—Precipitation after cell fragmentation

    表  1  Plackeet-Burman实验设计因素水平表

    Table  1.   Levels of selected factors for Plackeet-Burman

    FactorNameMass concentration/(g·L−1)
    Low level(−1)High level(+1)
    AGlucose0.51
    BYeast extract34
    CTryptone1015
    DKH2PO42.654
    ENaCl1015
    FVirtual variable
    GZn2+0.30.4
    HCa2+0.50.7
    I(NH4)2SO456.5
    JMn2+0.150.2
    KFe3+0.30.375
    LCo2+0.070.0875
    MMg2+45
    NVirtual variable
    下载: 导出CSV

    表  2  Plackeet-Burman实验设计及结果

    Table  2.   Experimental design and results of Plackeet-Burman

    RunFactorOD600Expression level/%OD600 × Expression level
    ABCDEFGHIJKLMN
    11−111−1−1−1−11−11−1115.1632.6168.053
    211−111−1−1−1−11−11−115.4736.05197.194
    3−111−11−1−1−1−11−11−114.833.00158.400
    4−1−111−111−1−1−1−11−114.5242.85193.682
    51−1−111−111−1−1−1−11−13.78534.95132.286
    611−1−111−111−1−1−1−114.43536.70162.765
    7111−1−111−111−1−1−1−15.47529.25160.144
    81111−1−111−111−1−1−15.8629.05170.233
    9−11111−1−111−111−1−15.0429.00146.144
    101−11111−1−111−111−14.5737.9170.233
    11−11−11111−1−111−1114.25536.45146.160
    121−11−11111−1−111−115.11528.65173.203
    13−11−11−11111−1−111−14.7133.05155.666
    14−1−11−11−11111−1−1114.2336.80155.664
    15−1−1−11−11−11111−1−114.4138.95171.769
    16−1−1−1−11−11−11111−1−14.0433.05133.522
    171−1−1−1−11−11−11111−14.8139.85191.679
    1811−1−1−1−11−11−111114.21537.10156.376
    19−111−1−1−1−11−11−11114.59533.15152.324
    20−1−1−1−1−1−1−11−1−1−1−1−1−14.39534.75152.726
    下载: 导出CSV

    表  3  Plackett-Burman实验回归分析结果

    Table  3.   Regression analysis results of Plackett-Burman design

    FactorNameFProb>F
    OD600Expression levelOD600 × Expression levelOD600Expression levelOD600 × Expression level
    AGlucose18.0933.60226.840.02380.00440.0006
    BYeast extract411.4076.89246.460.00030.00090.0006
    CTryptone314.7441.29502.470.00040.00300.0002
    DKH2PO4104.1259.20609.570.00200.00150.0001
    ENaCl4.1406.14895.990.13470.0684<0.0001
    FVirtual variable
    GZn2+3.73014.151121.790.14890.0197<0.0001
    HCa2+71.956.28673.430.00340.06640.0001
    I(NH4)2SO4110.0929.13345.750.00080.00550.0003
    JMn2+193.879.69939.010.00030.0358<0.0001
    KFe3+361.4923.13342.700.00050.00860.0003
    LCo2+280.9814.68190.710.15470.01860.0008
    MMg2+3.5820.38949.330.00270.0107<0.0001
    NVirtual variable
    R299.72%97.92%99.91%98.43%91.16%99.51%
    Relative deviation0.00210.00980.0004
    下载: 导出CSV
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出版历程
  • 收稿日期:  2020-08-24
  • 网络出版日期:  2020-10-28
  • 刊出日期:  2021-10-11

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