title: [Cell penetration of supercharged green fluorescent protein +36GFP as DNA carrier].
abstract: In this study, we expressed and purified supercharged green fluorescent protein (+36GFP) that we used to study its combination with nucleic acid and its cell transduction efficiency as carrier of DNA. We transformed pET+36GFP-HA2 plasmid into Escherichia coli BL21 (DE3), then expressed and purified the target protein. We used the protein to transduce a variety of mammalian cell lines including B16 cells, 293 cells, A549 cells and HepG2 cells at specified protein concentrations. Transduction efficiency of the protein was analyzed by flow cytometry. Under laser scanning confocal microscope, we observed visually transduction efficiency of +36GFP protein (100 nmol/L) to A549 cells. We incubated +36GFP with plasmid DNA and analyzed their binding ability with gel mobility shift assay. Then we transduced cells with the mixture of plasmid DNA/+36GFP protein at various ratio and detected the expression of reporter gene by using laser scanning confocal microscope and flow cytometry. The experimental results demonstrate that +36GFP had high transduction efficiency, and as the concentration increased, the efficiency improved in a dose-dependent manner. Gel mobility shift assay indicates that +36GFP could bind to plasmid DNA, blocking the migration of DNA in the gel in a concentration-dependent manner. After the plasmid wrapped by +36GFP penetrated into cells, the cells could express target protein efficiently, proving that +36GFP had the ability to carry nucleic acids into cells. Sucussful expression and purification of +36GFP protein confirms its high efficiency of cell transduction and its ability as carrier to deliver exogenous nucleic acids into cells.
authors.affiliation: Institute of Materia Medica, Zhejiang Academy of Medical Sciences, Hangzhou 310013, Zhejiang, China.
authors.forename: Hongyu
authors.initials: H
authors.lastname: Li
authors.forename: Yourong
authors.initials: Y
authors.lastname: Fang
authors.forename: Haitao
authors.initials: H
authors.lastname: Yu
authors.forename: Ying
authors.initials: Y
authors.lastname: Yu
authors.forename: Hui
authors.initials: H
authors.lastname: Yan
mesh_terms: D045744:Cell Line, Tumor; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D014161:Transduction, Genetic / Q000379:methods*; D014162:Transfection
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004247:DNA