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| The Optimization,Purification and Activity Detection of the Cell Penetrating Recombinase Tat-FLPo |
| WANG Qi1,2, YU Hui-qing1, CHEN Jian-quan1, ZENG Xian-yin2, CHENG Guo-xiang1 |
1. Shanghai Transgenic Research Center, Shanghai 201210, China;
2. Atomic Energy Application Laboratory, Sichuan Agriculture University, Ya’an 625014, China |
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Abstract In order to obtain the cell permeable recombinase Tat-FLPe for recombinant DNA research, a highly efficient and stable Tat-FLPe prokaryotic expression and purification system was established. First, the FLPe sequence was amplified by using the plasmid pCAGGS-FLPe as template, and then inserted FLPe into prokaryotic expression vector pET28a-Tat to get vector pET28a-Tat-FLPe for expression. Tat-FLPe were cloned into another three prokaryotic expression vector pET22b, pET30a, pET32a for expression respectively. As a result, only vector pET32a-Tat-FLPe induced stable Tat-FLPe expression in transformed Rosetta cells, while other three vectors failed to express or express at trace level. Further, in order to improve the expression level of Tat-FLPe, the composed codon of FLPe was optimized using online software. The expression level of optimized Tat-FLPe was increased significantly compared to the original one. On the other hand, the inducible conditions which could affect Tat-FLPe expression were explored, and found that the optimal induction condition of transformed cells was 0.05mol/L IPTG, 30 ℃, incubated for 4 hours. Finally, the expressed Tat-FLPe in Rosetta cells was purified by cation exchange column, the activity of cell permeable TAT-FLPe was verified by plasmid digestion experiments in vitro and cell experiments in vivo. In summary, the biological active Tat-FLPe recombinase in prokaryotic expression system were expressed successfully, thus laid a sound foundation for its application in genetic manipulation of cells and living animals.
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Received: 15 April 2013
Published: 25 August 2013
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