title: TAT-RhoGDI2, a novel tumor metastasis suppressor fusion protein: expression, purification and functional evaluation.
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.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, 200237, Shanghai, People's Republic of China.
authors.forename: Rui
authors.initials: R
authors.lastname: Xu
authors.forename: Yuguo
authors.initials: Y
authors.lastname: Dong
authors.forename: Lihua
authors.initials: L
authors.lastname: Wang
authors.forename: Xinyi
authors.initials: X
authors.lastname: Tao
authors.forename: Aiyou
authors.initials: A
authors.lastname: Sun
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
mesh_terms: D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects*; D003001:Cloning, Molecular; D004847:Epithelial Cells / Q000187:drug effects*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D062949:rho Guanine Nucleotide Dissociation Inhibitor beta / Q000235:genetics / Q000378:metabolism*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
chemical_list: C569658:ARHGDIB protein, human; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D062949:rho Guanine Nucleotide Dissociation Inhibitor beta; D054322:tat Gene Products, Human Immunodeficiency Virus