Title:The Efficiency of Tat Cell Penetrating Peptide for Intracellular Uptake of HIV-1 Nef Expressed in E. coli and Mammalian Cell
Volume: 14
Issue: 4
Author(s): Somayeh Kadkhodayan, Azam Bolhassani*, Seyed Mehdi Sadat, Shiva Irani and Fatemeh Fotouhi
Affiliation:
- Department of Hepatitis and AIDs Pasteur Institute of Iran, Tehran,Iran
Keywords:
Delivery systems, Eukaryotic and prokaryotic expression systems, HIV-1, Nef, Tat.
Abstract: 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 Tat-
Nef 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 ~ 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.
