文献类型：期刊文献

英文题名：TAT-RhoGDI2, a novel tumor metastasis suppressor fusion protein: expression, purification and functional evaluation

作者：Xu, Rui[1];Dong, Yuguo[1];Wang, Lihua[1];Tao, Xinyi[1];Sun, Aiyou[1];Wei, Dongzhi[1]

机构：[1]E China Univ Sci & Technol, New World Inst Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2014

卷号：98

期号：23

起止页码：9633

外文期刊名：APPLIED MICROBIOLOGY AND BIOTECHNOLOGY

收录：;EI(收录号：20144800238454);WOS:【SCI-EXPANDED(收录号:WOS:000345140200009)】；

基金：This work was supported by the grant from the National Major Science and Technology Projects of China (Grant No. 2012ZX09304009).

语种：英文

外文关键词：RhoGDI2; TAT-fused protein; Purification; Bladder cancer; Metastasis

摘要：Rho GDP dissociation inhibitor 2 (RhoGDI2) was identified as a functional metastasis suppressor in human bladder cancer, suggesting that increasing the RhoGDI2 level may represent a promising therapeutic strategy. It has been shown that the transactivator of transcription (TAT) protein from HIV-1 is able to efficiently deliver various biological molecules into several cell types. In this study, TAT peptide was fused with the N-terminus of RhoGDI2, and the resulting TAT-RhoGDI2 fragment was inserted into the pGEX-6p-1 plasmid and expressed as a glutathione S-transferase (GST)/TAT-RhoGDI2 fusion protein in Escherichia coli BL21(DE3) cells. A two-step purification strategy involving glutathione sepharose chromatography and PreScission protease cleavage was developed to purify TAT-RhoGDI2; subsequently, the identification of the involved macromolecules was achieved by Western blot. The final product, TAT-RhoGDI2, was obtained at a concentration of 112 mg/L. This is the first report on the efficient production of bioactive TAT-RhoGDI2 through a gene-engineering approach in E. coli. Using flow cytometry, we found that the TAT-RhoGDI2 fusion proteins could penetrate into bladder cancer cells with an extremely high efficiency. In vitro scratch and transwell assay and the migration/invasion behavior of UMUC3 cells were strongly reduced by the treatment with TAT-RhoGDI2. These studies support the use of the TAT-RhoGDI2 protein in tumor metastasis therapy.