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| Preparation of Recombinant PTD-KLF4 with Penetrating Peptide and Activity Assay |
| SU Hang1, YU Rong-jie3, LIU Xiao-fei3, WANG Jing-jing3, LI Xiao-xia2, CHEN Jian-su1,2 |
1. Key Laboratory for Regenerative Medicine, Ministry of Education, Jinan University, Guangzhou 510632, China;
2. Ophthalmology Department,Medical College, Jinan University, Guangzhou 510632, China;
3. Bioengineering Institute of Jinan University, Guangzhou 510632, China |
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Abstract KLF4 is one member of the kruppel-like factors (KLF) family, which is an important transcription factor to maintain the pluripotency of embryonic stem cells (ESCs). Protein transduction domain (PTD) can carry macromolecules into cells. In order to obtain the recombinant KLF4 protein with penetrating membrane activity,the KLF4 gene was expressed as a fusion protein PTD-KLF4 combined with PTD using prokaryotic expression vector PKYB. The fusion protein PTD-KLF4 was purified by Ni affinity chromatography and identified by Western blotting analysis. The fluorescein isothiocyanate (FITC) labeled PTD-KLF4 was used to investigate the penetrating ability of the fusion protein into Chinese hamster ovary (CHO) cells. And the bioactivity of PTD-KLF4 binding the target DNA sequence was detected by fluorescence resonance energy transfer (FRET). The results showed that the recombinant PTD-KLF4 successfully entered the cells and located in the nucleus with the transmembrane efficiency of (22.29±2.1)%.The recombinant PTD-KLF4 caused cell shape changes and had specific binding capacity with its target DNA sequence.The preparation of the recombinant PTD-KLF4 laid a good foundation for the induction of pluripotent stem cells (IPSCs) with exogenous protein.
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Received: 09 May 2011
Published: 25 September 2011
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