TAT-RhoGDI2, a novel tumor metastasis suppressor fusion protein: expression, purification and functional evaluation.
Appl Microbiol Biotechnol
; 98(23): 9633-41, 2014 Dec.
Article
em En
| MEDLINE
| ID: mdl-25142698
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Movimento Celular
/
Proteínas Supressoras de Tumor
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Células Epiteliais
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Produtos do Gene tat do Vírus da Imunodeficiência Humana
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Inibidor beta de Dissociação do Nucleotídeo Guanina rho
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
Appl Microbiol Biotechnol
Ano de publicação:
2014
Tipo de documento:
Article