A Novel DNA Delivery System Mediated By Cell-penetrating Peptides

DNA vaccines have potential application for the emergent control of the outbreak of infectious diseases. However, the low efficiency of null DNA in cellular transfection, consequently the low expression level of its encoding antigen in vivo, represents the major problem for preventing its usage as an effective vaccine form at present. In this present study, we explore the possibility of the fusion protein of cell-penetrating peptides and LacI headpiece mutant as a carrier for improving the cellular transfection efficiency of null DNA, thereby providing a novel universal delivery system for DNA vaccines.Cell-penetrating peptides (CPPs) is a class of small molecular peptides with the ability of high efficient cellular penetration. The currently identified CPPs include HIV TAT, ANTP, VP22, et al. It has been demonstrated that CPPs can mediate the delivery of various molecules including proteins and DNAs into various cell types without the cell damaging effects, and therefore have important application in drug delivery.CPPs used in this research are ANTP and TAT. ANTP is the thirdα-helix in the homeodomain of Drosphila Antennapedia, and is also the first reported CPPs used in exogenous cargo transduction. TAT is derived from a section of the peptide sequence of trans-activating transcriptional activator of human immunodeficiency virus (HIV-1) and is one of the most intensely investigated CPPs at present.LacI is the transcriptional repressor of the Lac gene in bacteria and can specifically bind to its recognizing DNA sequence with high affinity. The DNA binding domain of LacI is within its extreme N-terminal amino acid sequence (aa1-357). It has recently demonstrated that this N-terminal peptide sequence alone, when dimerization, possess the same and even higher affinity for the Lac DNA sequence as the full-length LacI protein and has termed as the LacI headpiece mutant(LacI HPM, LacI HP62-V52C).To construct CPP-LacI HPM fusion proteins, the DNA sequence encoding the LacI, LacI HP and LacI HPM were firstly amplified by PCR and fused in-framed with DNA sequence encoding TAT, TAT-RGD, ANTP, and ANTP-RGD, respectively. These resulted fusion genes were individually cloned into either pET-28a(+) or pGEX-KG plasmids and transformed into BL21 Star Escherichia coli for expression.Fusion proteins produced in E. coli were purified by either Ni-NTA or Glutathione Sepharose 4B Beads collumns, and then were dialyzed at room temperature against buffer to form the CCP-LacI HPM dimers. After dialyzation, samples were concentrated by PEG-8000.We examined the DNA-binding activity of the produced CCPs-LacI HPM dimer by its incubation with tested plasmids which contains the LBS (LacI specific binding sequence). After intensively washing, the protein sample were subjected to PCR assay for the dectection of its binding DNA and confirmed that the desigend CCPs-LacI HPM dimer have high affinty for its recognizing DNA sequences.Immunofluorescence studies were carried out to identify transduced dimeric CPPs-LacI HPM fusion proteins could penetrate in cells. Then, Monolayer cultured cells were disrupted after the test plasmid transduction mediated by the dimeric fusion proteins. Western blotting was performed as demonstrated proteins localized in transfected cells.We further demonstrated that our designed CPPs-LacI HPM fusion proteins can mediate the reporter EGFP gene transfection and expression in human cells. Therefore, our data suggest the CPPs-LacI HPM fusion proteins may have the potential to serve as a novel safe, efficient DNA delivery system which may improve the application of DNA vaccines in the future.