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  • CN 31-1691/TQ

赖氨酰内切酶在大肠杆菌中的重组表达、复性及纯化

朱奕 徐名强 任燕娜 蔡孟浩

朱奕, 徐名强, 任燕娜, 蔡孟浩. 赖氨酰内切酶在大肠杆菌中的重组表达、复性及纯化[J]. 华东理工大学学报(自然科学版). doi: 10.14135/j.cnki.1006-3080.20220607001
引用本文: 朱奕, 徐名强, 任燕娜, 蔡孟浩. 赖氨酰内切酶在大肠杆菌中的重组表达、复性及纯化[J]. 华东理工大学学报(自然科学版). doi: 10.14135/j.cnki.1006-3080.20220607001
ZHU Yi, XU Mingqiang, REN Yanna, CAI Menghao. Recombinant Expression, Refolding, and Purification of Lysobacter enzymogenes Lys-C in Escherichia coli[J]. Journal of East China University of Science and Technology. doi: 10.14135/j.cnki.1006-3080.20220607001
Citation: ZHU Yi, XU Mingqiang, REN Yanna, CAI Menghao. Recombinant Expression, Refolding, and Purification of Lysobacter enzymogenes Lys-C in Escherichia coli[J]. Journal of East China University of Science and Technology. doi: 10.14135/j.cnki.1006-3080.20220607001

赖氨酰内切酶在大肠杆菌中的重组表达、复性及纯化

doi: 10.14135/j.cnki.1006-3080.20220607001
详细信息
    作者简介:

    朱奕:朱 奕(1996-),男,浙江宁波人,硕士生,主要研究方向:蛋白药物制备。E-mail:zhuyi0304@163.com

    通讯作者:

    任燕娜, E-mail:15026455240@163.com

    ;蔡孟浩, E-mail:cmh022199@ecust.edu.cn

  • 中图分类号: Q556

Recombinant Expression, Refolding, and Purification of Lysobacter enzymogenes Lys-C in Escherichia coli

  • 摘要: 在产酶溶杆菌来源的Lys-C成熟肽序列的N端和C端分别融合人工前肽 (MGSK) 和6×His标签,将密码子优化后的序列插入表达载体pET-28a。以IPTG诱导型启动子PT7控制Lys-C的高效表达,并针对重组菌株JM109DE3_PT7-LysC开展生物反应器高密度发酵生产Lys-C。然后收集和溶解包涵体得到Lys-C变性液,通过Sephadex G25层析脱除DTT,并在Lys-C复性液中添加前导肽 (pre-N-pro) 辅助成熟肽蛋白折叠。进一步,通过切向流过滤、Ni NTA-Sepharose亲和层析和Sephacryl S-100层析等一系列纯化步骤,获得高纯度的重组Lys-C。最后进行酶切三肽底物和门冬胰岛素前体检测,分析重组Lys-C的活性水平。重组Lys-C发酵产量为2.4 g/L,经复性和纯化后的终产量可达48 mg/L。添加80 mg/L pre-N-pro促进了重组Lys-C的复性,复性后酶活相比于未添加pre-N-pro时提升了4.8倍,最高可达到13.8 AU/L。经过多步纯化后,重组Lys-C的比酶活为10.2 AU/mg,且对于门冬胰岛素前体的酶切转化率可达93.5%。

     

  • 图  1  Lys-C和pre-N-pro的基因与蛋白序列

    Figure  1.  The gene and protein sequences of Lys-C and pre-N-pro

    图  2  质粒pET28a-LysC和pET28a-preNpro的构建与验证

    Figure  2.  The Construction and verification of plasmids pET28a-LysC and pET28a-preNpro

    图  3  LysC和pre-N-pro的诱导表达

    Figure  3.  The inducible expression of LysC and pre-N-pro

    图  4  Lys-C的复性

    Figure  4.  Refolding of LysC

    图  5  Lys-C的纯化

    Figure  5.  Purification of Lys-C

    图  6  Lys-C酶切胰岛素前体的HPLC图谱

    Figure  6.  The HPLC chromatograms of the insulin precursor digested by recombinant Lys-C

    表  1  本研究使用引物

    Table  1.   Primers used in this study

    PrimersSequence (5’ to 3’)
    T7lacO-F CACCATTAAGATCCGGCTGCTAAC
    T7lacO-R CCATGGTATATCTCCTTCTTAAAG
    T7lacOLysC-F AAGAAGGAGATATACCATGGGCAGCAAAGGTGTGAG
    T7lacOLysC-R GTTAGCAGCCGGATCTTAATGGTGGTGATGATGGTGAACTGG
    28aarmpNp-F TAAGAAGGAGATATACCATGAAACGCATTTGCGGTAG
    pNp28ahisarm-R TTAATGGTGGTGATGATGGTGTTTTTCGCCGCTGGCGG
    V28aUP-F GGAAGCAGCCCAGTAGTAGG
    VT7DO-R CAAGACCCGTTTAGAGGCCC
    V28aDO-R CGATGGCCCACTACGTGAAC
    下载: 导出CSV

    表  3  HPLC洗脱程序

    Table  3.   HPLC elution procedure

    Time/minAB
    062%38%
    2054%46%
    2554%46%
    2810%90%
    3262%38%
    40Stop Stop
    下载: 导出CSV

    表  2  比色法测酶活的操作方法

    Table  2.   Method for measuring Lys-C activity by chromogenic reaction using Bz-Lys-pNA as a substrate

    ReagentsControlLys-C sample
    A200 μL200 μL
    B20 μL20 μL
    30℃ water bath, preheat for 5 min
    C10 μL--
    D--10 μL
    Mix quickly, 30℃ water bath for 30 min
    E70 μL70 μL
    下载: 导出CSV

    表  4  Lys-C各步纯化结果

    Table  4.   Lys-C purification results of each step

    StepsVolume/mLProtein amount/mgRecovery/%
    Cell lysis--7600--
    Washing--2400--
    Denaturation130640--
    GFC (Sephadex G25)27055085.9
    TFF25043078.2
    NCAC409522.1
    UF205760.5
    GFC (Sephacryl S-100)424884.8
    下载: 导出CSV

    表  5  大肠杆菌重组表达Lys-C的产量结果

    Table  5.   Yield analysis of recombinant expression of Lys-C in E. coli.

    SourceHostPlasmidYieldLiterature
    AchromobacterE. coli lon-pKK233-20.48 mg/L1989,Ohara[21]
    AchromobacterE. coli JA221pKYN2000.3~0.4 mg/L2002,Kentaro[8]
    LysobacterE. coli JM109pGEM-T11 mg/L2002,Shigeru[11]
    LysobacterE. coli JM109pJOE24 mg/L2016,Stressler[22]
    PseudomonasE. coli BL21(DE3)pET-32a34.4 mg/L2016,Zhao[23]
    LysobacterE. coli JM109(DE3)pET-28a48 mg/LThis study
    下载: 导出CSV
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出版历程
  • 收稿日期:  2022-06-07
  • 网络出版日期:  2022-08-26

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