Design and construction of an effective expression system with aldehyde tag for site-specific enzyme immobilization

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  • Fang Wang, Jilin University
  • ,
  • Rong Li
  • Hui Jian, Jilin University
  • ,
  • Zihao Huang, Jilin University
  • ,
  • Yingwu Wang, Jilin University
  • ,
  • Zheng Guo
  • Renjun Gao, Jilin University

In recent years, the development and application of site-specific immobilization technology for proteins have undergone significant advances, which avoids the unwanted and random covalent linkage between the support and active site of protein in the covalent immobilization. Formylglycine generating enzyme (FGE) can transform the cysteine from a conversed 6-amino-acid sequence CXPXR into formylglycine with an aldehyde group (also termed as “aldehyde tag”). Based on the frame of pET-28a, the His-tags were replaced with aldehyde tags. Afterward, a set of plasmids were constructed for site-specific covalent immobilization, their His-tags were knock out (DH), or were replaced at different positions: N-terminal (NQ), C-terminal (CQ), or both (DQ) respectively. Three different enzymes, thermophilic acyl aminopeptidase (EC from Sulfolobus tokodaii (ST0779), thermophilic dehalogenase (EC from Sulfolobus tokodaii (ST2570), and Lipase A (EC from Bacillus subtilis (BsLA) were chosen as model enzymes to connect with these plasmid systems. The results showed that different aldehyde-tagged enzymes can be successfully covalently attached to different carriers modified with an amino group, proving the universality of the method. The new immobilized enzyme also presented better thermostability and reutilization than those of the free enzyme.

Antal sider14
StatusUdgivet - 2020

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