The Efficiency of Tat Cell Penetrating Peptide for Intracellular Uptake of HIV-1 Nef Expressed in E-coli and Mammalian Cell

作者：Kadkhodayan Somayeh; Bolhassani Azam^*; Sadat Seyed Mehdi; Irani Shiva; Fotouhi Fatemeh

来源：Current Drug Delivery, 2017, 14(4): 536-542.

DOI：10.2174/1567201813666161006114448

• Delivery systems
• Eukaryotic and prokaryotic expression systems
• HIV-1
• Nef
• Tat

Background: Cell penetrating peptides (CPPs) or protein transduction domains (PTDs) have been known as a new field in cargo delivery. These peptides such as Tat, Pep-1 and Cady-2 are able to deliver genes and biologically active proteins to cytoplasmic compartments via the plasma membrane.
Methods: In current study, the efficiency of pEGFP-N1 eukaryotic vector for expression of HIV-1 TatNef fusion was evaluated in HEK-293T cells using TurboFect transfection reagent. In addition, the recombinant GST-Tat-Nef protein was generated in E. coli and transfected using two amphipathic CPPs (Pep-1 and Cady-2) into mammalian cells. The size and morphology of the CPP/GST-Tat-Nef complexes were evaluated by scanning electron microscopy (SEM), Zetasizer, and SDS-PAGE. The transfection of HIV-1 GST-Tat-Nef protein was also analyzed using SDS-PAGE and western blotting.
Results: Our data indicated that the recombinant GST-Tat-Nef protein generated in BL21 strain migrated as a clear band of similar to 52 kDa in SDS-PAGE. The results of SEM and Zetasizer confirmed the formation of protein/Pep-1 or protein/Cady-2 nanoparticles less than 200 nm in diameter. Tat CPP fused to Nef protein could deliver the recombinant Nef protein alone and notably by forming the noncovalent complexes with TurboFect, Pep-1 and Cady-2 as detected in western blotting. Moreover, intracellular uptake of Tat-Nef gene and subsequently its expression in mammalian cells was considerably higher than that for Nef gene.
Conclusion: This data indicated that the Tat gene sequence could also increase the transfection of Nef gene in vitro. Generally, the Tat-Nef interaction led to enhance further gene expression and also protein delivery.

• 出版日期2017