title: The Efficiency of Tat Cell Penetrating Peptide for Intracellular Uptake of HIV-1 Nef Expressed in E. coli and Mammalian Cell.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biology, School of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, Iran.
authors.forename: Somayeh
authors.initials: S
authors.lastname: Kadkhodayan
authors.affiliation: Department of Hepatitis and AIDs Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Azam
authors.initials: A
authors.lastname: Bolhassani
authors.affiliation: Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Seyed Mehdi
authors.initials: SM
authors.lastname: Sadat
authors.affiliation: Department of Biology, School of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, Iran.
authors.forename: Shiva
authors.initials: S
authors.lastname: Irani
authors.affiliation: Influenza Research Lab., Department of Virology, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Fotouhi
mesh_terms: D000818:Animals; D057846:Cell-Penetrating Peptides / Q000737:chemistry*; D004926:Escherichia coli; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000235:genetics*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics*
chemical_list: D057846:Cell-Penetrating Peptides; D011993:Recombinant Fusion Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1; D054322:tat Gene Products, Human Immunodeficiency Virus
keywords: Delivery systems; Eukaryotic and prokaryotic expression systems; HIV-1; Nef; Tat