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

Content Provider	Springer Nature Link
Author	Xu, Rui Dong, Yuguo Wang, Lihua Tao, Xinyi Sun, Aiyou Wei, Dongzhi
Copyright Year	2014
Abstract	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.
Starting Page	9633
Ending Page	9641
Page Count	9
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	98
Issue Number	23
e-ISSN	14320614
Language	English
Publisher	Springer Berlin Heidelberg
Publisher Date	2014-08-22
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	RhoGDI2 TAT-fused protein Purification Bladder cancer Metastasis Microbiology Microbial Genetics and Genomics Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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