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Archive > Volume 20 Issue 2 > 2013,20(2):201-206. DOI:10.3872/j.issn.1007-385X.2013.02.013 Prev Next
Basic Research
Prokaryotic expression of 3TAT-DRBD fusion protein and identification of itssiRNA-binding activity and membrane-penetrating function
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Abstract:
Objective: To construct a recombinant vector 3TAT-DRBD expressing purified fusion protein, and to preliminary validate its siRNA-binding activity and membrane-penetrating function. Methods: The target gene 3TAT-DRBD was obtained by gene synthesis and cloned to prokaryotic expression vector pET-44b. The expression of fusion protein was induced by IPTG. The fusion protein was purified by Ni-NTA agarose, and cut by thrombin and detected by Western blotting analysis. The binding activity of DRBD was tested by EMSA and the cytomembrane penetrating activity of TAT was observed by confocal laser scanning microscopy (CLSM). Results: Restriction enzyme digestion and gene sequencing showed that the recombinant plasmid pET-44b-3TAT-DRBD was successfully constructed. The fusion protein (containing Nus and S tags) induced by IPTG was efficiently expressed in E. coli, with the soluble parts accounting for around 80% of the total proteins. The tags were successfully cut off and the fusion protein without tags was purified with a molecular weight of 17 000 Da identified by Western blotting. EMSA identified that the fusion protein 3TAT-DRBD could effectively bind siRNA targeting survivin gene (survivin-siRNA). The efficiency of survivin-siRNA penetrating into prostate cancer PC3 cells mediated by TAT was significantly increased under an observation of CLSM. Conclusion: 3TAT-DRBD fusion protein with siRNA-binding activity and cell membrane-penetrating function is successfully expressed and purified, lying a good basis for further functional research and clinical application of 3TAT-DRBD.
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Project Supported:
Project supported by the National Natural Science Foundation of China（No.30973463，No.81172446）
Clc Number:
