肽脱甲酰基酶在偶极离子修饰亲水磁珠表面的固定化及表征  

Immobilization and characterization of peptide deformylase on surface of ampholytic ion modified hydrophilic magnetic beads

作　　者：李欣蓬 杨林玉 段昌园 杨晓兰[1] LI Xinpeng;YANG Linyu;DUAN Changyuan;YANG Xiaolan(Key Laboratory of Clinical Laboratory Diagnostics of Ministry of Education,College of Laboratory Medicine,Chongqing Medical University,Chongqing,400016,China)

机构地区：[1]重庆医科大学检验医学院,临床检验诊断学教育部重点实验室,重庆400016

出　　处：《第三军医大学学报》2021年第6期481-488,共8页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(31570862,81773625)。

摘　　要：目的比较表面修饰偶极离子且带Ni^(2+)-NTA或羧基的两种亲水磁珠对结核分枝杆菌肽脱甲酰基酶(Mycobacterium tuberculosis peptide deformylase,MtPDF)的固定化效果,探讨适于磁分离联用HPLC-MS筛选MtPDF配体混合物库的靶酶固定化方案。方法将N端带有6×His标签MtPDF的质粒pET-28a(+)导入E.coli BL21(DE3)重组表达,使用镍离子螯合亲和层析介质纯化后表征其酶学性质。将MtPDF与Ni^(2+)-NTA或羧基磁珠偶联并比较其固定化容量、保留活性、固定化前后酶稳定性及对抑制剂放线酰胺素(Actinonin)的响应。结果 Ni^(2+)-NTA磁珠固定MtPDF的容量为(99.7±4.5) mg/g (n=3);最优投料比酶量∶磁珠量=1∶15时,固定化酶保留活性为(107.2±13.9)%(n=3),在0℃、pH=7.5缓冲液中振摇3 h活性仅保留43%。羧基磁珠活化后通过酰胺键共价固定MtPDF的容量为(36.9±2.6) mg/g (n=3);最优投料比酶量∶磁珠量=1∶60时,固定化酶保留活性为(102.6±15.4)%(n=3),与Ni^(2+)-NTA磁珠固定化保留活性相当;在0℃、pH 7.5缓冲液中振摇8 h活性无显著改变;羧基磁珠固定化酶对Actinonin的抑制响应与游离酶无显著差异(P>0.05)。结论羧基磁珠固定化MtPDF酶活更稳定,不影响抑制剂响应,适于混合物库中MtPDF高亲和力配体筛选。Objective To compare the immobilization effect of 2 kinds of hydrophilic magnetic beads with Ni^(2+)-nitrilotriacetic acid(Ni^(2+)-NTA) group or carboxyl group and surface modified by ampholytic ions on Mycobacterium tuberculosis peptide deformylase(MtPDF), and to determine the target enzyme immobilization scheme appliable to screening MtPDF ligand mixture library using magnetic separation combined with high performance liquid chromatography-mass spectrometry(HPLC-MS). Methods The plasmids pET-28 a(+): def with N-terminal 6×His tagged MtPDF was successfully constructed and transfected into E.coli BL21(DE3) for MtPDF recombinant expression. The products were collected and purified using nickel-ion chelating affinity chromatography column, and then identified with the enzymatic properties. Then the obtained MtPDF was coupled to magnetic beads with Ni^(2+)-NTA(MtPDF-Ni^(2+)-NTA) or carboxyl group(MtPDF-carboxy), respectively. The immobilization capacity, enzyme activity, enzyme stability before and after immobilization, and the response to the inhibitor actinonin were compared between the 2 schemes. Results The immobilization capacity of MtPDF-Ni^(2+)-NTA was 99.7±4.5 mg/g(n=3). When the ratio of amount of enzyme to magnetic beads was 1∶15, the retention activity of the immobilized MtPDF was(107.2±13.9)%(n=3) of the free enzyme, and decreased to 43% after 3 h under shaking at 0 ℃ and pH value of 7.5. By contrast, the immobilization capacity of MtPDF-carboxy was 36.9±2.6 mg/g(n=3), and the retention activity of the immobilized MtPDF reached to(102.6±15.4)%(n=3) when the ratio of amount of enzyme to magnetic beads was 1∶60, consistent with that on the Ni^(2+)-NTA magnetic beads, with negligible changes within 8 h under shaking at 0 ℃ and pH 7.5. The response of MtPDF-carboxy to the inhibition of actinonin had no significant difference from that of free MtPDF(P>0.05). Conclusion MtPDF-carboxy magnetic beads present stable retention activity, and has similar inhibitory response as that of free MtPDF, and th

关 键 词：磁珠固定化 肽脱甲酰基酶 结核 筛选 

分 类 号：R378.91[医药卫生—病原生物学] R931.6[医药卫生—基础医学]