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Reconstruction of enzymatic activity from split genes encoding glyphosate-tolerant EPSPS protein of Psedomonas fluorescens G2 strain by intein mediated protein complementation

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Chinese Science Bulletin

Abstract

A mutagenesis library was constructed using GPS-LS system to insert a random 5 aa into the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoded by aroA gene. Active EPSPS proteins were identified by the ability to rescue growth of aroA-deleted mutant ER2799 on M9 minimal media. 12 unique sites, which can tolerate a 5-aa insertion, were identified. In all of the 12 sites, only F295/T296 site was found to split the G2-EPSPS properly by co-transformation of plasmids into E. coli ER2799. The G2-EPSPS gene was then divided into N-terminal and C-terminal from F295/T296 site which were fused to the N-terminal and C-terminal of Ssp.DnaE intein, respectively, creating two plasmids pMEPSN295IN and pKEPSc296lc. Co-transformation of plasmids, pMEPSN295IN and pKEPSc296lc, rescued growth of ER2799 in M9 minimal media, indicating that the intein splicing domains were bringing the EPSPS fragments together to generate activity. Reconsituted activity of splitted G2-EPSPS enzyme was 4.48 U/mg.

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Authors and Affiliations

  1. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China

    Dun Baoqing, Lu Wei, Zhang Wei, Ping Shuzhen, Wang Xujing, Chen Ming, Xu Yuquan, Jin Dan, Wang Jin, Zhao Zhonglin, Liang Aimin, Hou Songna & Lin Min

  2. New England Biolabs, Inc., Beverly, Massachusetts, 01915, USA

    Xu Ming-Qun

Authors
  1. Dun Baoqing
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  2. Lu Wei
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  3. Zhang Wei
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  4. Ping Shuzhen
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  5. Wang Xujing
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  6. Chen Ming
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  7. Xu Yuquan
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  8. Jin Dan
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  9. Wang Jin
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  10. Zhao Zhonglin
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  11. Liang Aimin
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  12. Hou Songna
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  13. Xu Ming-Qun
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  14. Lin Min
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Corresponding author

Correspondence to Lin Min.

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Dun, B., Lu, W., Zhang, W. et al. Reconstruction of enzymatic activity from split genes encoding glyphosate-tolerant EPSPS protein of Psedomonas fluorescens G2 strain by intein mediated protein complementation. CHINESE SCI BULL 51, 1652–1654 (2006). https://doi.org/10.1007/s11434-006-2017-0

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  • DOI: https://doi.org/10.1007/s11434-006-2017-0

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