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.

Intranasal Delivery of Recombinant AAV Containing BDNF Fused with HA2TAT: a Potential Promising Therapy Strategy for Major Depressive Disorder

“…The adenovirus associated virus (AAV), a single-stranded DNA virus with an icosahedral capsid, is a promising vector for gene transferring in vivo 19 . The transactivator of transcription (TAT) protein from human immunodeficiency virus (HIV)-1, an 11-amino acid peptide, is an excellent candidate for protein transduction which could fuse with other proteins to improve their penetration 20 21 . The HA2, a subunit of the influenza A virus hemagglutinin glycoprotein HA, includes a hydrophobic peptide sequence which could facilitate lipid membrane destabilization 22 23 .…”

High yields and soluble expression of superoxide dismutases in Escherichia coli due to the HIV-1 Tat peptide via increases in mRNA transcription

“…25, 26 The core region of Tat comprises a basic amino acid domain that contains six arginines and two lysines. 27, 28, 29 The current study showed that the transmembrane transduction efficiency of heterologous proteins was immediately affected by the number of amino acids. The prediction of protein secondary structure results showed that the α-helix structure could be formed in the Tat basic amino acid domain.…”

Brain-Derived Neurotrophic Factor Delivered Intranasally Relieves Post-Traumatic Stress Disorder Symptoms Caused by a Single Prolonged Stress in Rats

“…HA2 is a subunit of the hemagglutinin glycoprotein protein HA of the influenza A virus, including a hydrophobic peptide segment, that promotes lipid membrane instability [14]. TAT is an 11-amino acid peptide fragment of HIV-1, has a protein-transport function, and can fuse to other proteins to increase penetrability [15,16]. In our previous study, we successfully constructed BDNF-HA2TAT/AAV [17,18].…”