| 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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| 作者姓名： | DUN Baoqing LU Wei ZHANG Wei PING Shuzhen WANG Xujing CHEN Ming XU Yuquan JIN Dan WANG Jin ZHAO Zhonglin LIANG Aimin HOU Songna XU Ming-Qun LIN Min |
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| 作者单位： | [1]Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China [2]New England Biolabs, Inc., Beverly, Massachusetts 01915, USA |
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| 基金项目： | Acknowledgements This work was supported by the National Project ＂973＂ （Grant No. 001CB108904） and the National High Technology Project ＂863＂ （Grant No. 2004AA214170）. |
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| 摘 要： | N-phosphonomethylglycine, commonly referred to as glyphosate, is a broad-spectrum, non-selective herbicide used for the control of weeds in glyphosate-to- lerant crops. Glyphosate inhibits the 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS), a key en…
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| 关 键 词： | 断裂基因 蛋白质再生 转基因 EPSPS |
| 收稿时间： | 2006-03-25 |
| 修稿时间： | 2006-03-252006-05-18 |
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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| DUN Baoqing LU Wei ZHANG Wei PING Shuzhen WANG Xujing CHEN Ming XU Yuquan JIN Dan WANG Jin ZHAO Zhonglin LIANG Aimin HOU Songna XU Ming-Qun LIN Min.Reconstruction of enzymatic activity from split genes encoding glyphosate-tolerant EPSPS protein of Psedomonas fluorescens G2 strain by intein mediated protein complementation[J].Chinese Science Bulletin,2006,51(13):1652-1654. |
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| Authors: | Baoqing Dun Wei Lu Wei Zhang Shuzhen Ping Xujing Wang Ming Chen Yuquan Xu Dan Jin Jin Wang Zhonglin Zhao Aimin Liang Songna Hou Ming-Qun Xu Min Lin |
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| Institution: | (1) Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China;(2) New England Biolabs, Inc., Beverly, Massachusetts 01915, USA |
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| 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 pro- teins 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-termi- nal 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 pMEPS- N295IN and pKEPSc296Ic. Co-transformation of plasmids, pMEPSN295IN and pKEPSc296Ic, rescu- ed growth of ER2799 in M9 minimal media, indicating that the intein splicing domains were bringing the EPSPS fragments together to generate activity. Re- consituted activity of splitted G2-EPSPS enzyme was 4.48 U/mg. |
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| Keywords: | splitting gene intein protein reconstitution transgene |
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