title: [Construction and functional study of a cell penetrating peptide-based expression vector for targeted delivery of proteins into the cell nuclei].
abstract: OBJECTIVE
  To construct an cell penetrating peptide-based expression vector capable of targeted delivery of proteins into the cell nuclei and study its function of protein transduction.
  
  
  METHODS
  The fusion protein expression vector pET14b-HC(L)NE (pET14-b-His-CPP-Linker-NLS-EGFP) incorporating cell penetrating peptide (CPP), nuclear localization signal(NLS), linker and enhanced green fluorescent protein (EGFP) was constructed based on His-tagged pET14b-HE (pET14b-His-EGFP) by site-directed mutagenesis PCR method. After identification by enzyme digestion and DNA sequencing, the recombinant plasmid was transformed into BL21(DE(3)) strain. The HC(L)NE fusion protein was expressed following IPTG induction and purified with Ni(2+)-NTA affinity chromatography. After dialysis and filtration, the HC(L)NE fusion protein was added into cultured eukaryotic cells. The protein transduction in the living cells was observed under fluorescence microscope and analyzed by Western blotting.
  
  
  RESULTS
  Enzyme digestion and DNA sequencing confirmed successful construction of the pET14b-HC(L)NE vector, and the fusion protein efficiently expressed in E. coli. Protein transduction experiments in eukaryotic cells revealed that the fusion protein could rapidly penetrate the cell membrane and reach the cell nucleus, and this internalization was time- and concentration-dependent.
  
  
  CONCLUSIONS
  The cell penetrating peptide-based expression vector for targeted protein delivery to the cell nucleus has been successfully constructed, and a transport system that can delivery exogenous proteins or polypeptides into the cytoplasm and cell nucleus is established, which provides an economical and efficient means for functional study of the proteins and polypeptide in cells and targeted drug delivery.
authors.affiliation: Department of Pathophysiology and Key Laboratory of Functional Proteomics of Guangdong Province, Southern Medical University, Guangzhou 510515, China. lhy245@fimmu.com
authors.forename: Hai-yu
authors.initials: HY
authors.lastname: Li
authors.forename: Ai-hua
authors.initials: AH
authors.lastname: Guo
authors.forename: Zhi-feng
authors.initials: ZF
authors.lastname: Liu
authors.forename: Yu
authors.initials: Y
authors.lastname: Liu
authors.forename: Jing-hua
authors.initials: JH
authors.lastname: Liu
authors.forename: Peng
authors.initials: P
authors.lastname: Deng
authors.forename: Zhi-jie
authors.initials: ZJ
authors.lastname: Li
authors.forename: Ya-wei
authors.initials: YW
authors.lastname: Liu
authors.forename: Yong
authors.initials: Y
authors.lastname: Jiang
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells; D019913:Nuclear Localization Signals / Q000235:genetics; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D019913:Nuclear Localization Signals; D010455:Peptides; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins