[Cell-penetrating peptide PEP-1-mediated transduction of enhanced green fluorescent protein into human umbilical vein endothelial cells].
OBJECTIVE
To investigate the penetrating ability of fusion protein PEP-1-EGFP with human umbilical vein endothelial cells.


METHODS
Two prokaryotic expression plasmids pET15b-EGFP and pET15b-PEP-1-EGFP were constructed and transformed into E. coli BL21 (DE3) to express EGFP and fusion protein PEP-1-EGFP, respectively. The expressed EGFP and PEP-1-EGFP were purified with Ni(2+) -resin affinity chromatography, and their capabilities of transduction into human umbilical vein endothelial cells were evaluated. The time- and dose-dependent transduction of the fusion protein PEP-1-EGFP and its stability in the human umbilical vein endothelial cells were observed. The toxicity of the fusion protein PEP-1-EGFP was detected by MTT method.


RESULTS
EGFP failed to be transduced into human umbilical vein endothelial cells, whereas PEP-1-EGFP fusion protein was transduced into cells shortly in 5 minutes. Its transduction was time- and dose-dependent and the fluorescence in the cells were detected even 27 hours later. No cytotoxicity of the fusion protein PEP-1-EGFP to human umbilical vein endothelial cells was detected even when the dose reached up to 200 micromol/L.


CONCLUSIONS
PEP-1-EGFP fusion protein can efficiently transduce the target protein into human umbilical vein endothelial cells, which provides a basis for future researches on the transduction of antioxidant enzymes mediated by the cell-penetrating peptide, PEP-1, in ischemia-reperfusion injury therapy.
Dong
Xiao
X
Institute of Clinical Medicine, Renmin Hospital, Yunyang Medical College, Hubei Shiyan 442000, China.
Wang
Jia-Ning
JN
Huang
Yong-Zhang
YZ
Guo
Ling-Yun
LY
Kong
Xia
X
D002478:Cells, Cultured; D003543:Cysteamine / Q000031:analogs & derivatives* / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D010455:Peptides / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000633:toxicity; D014471:Umbilical Veins / Q000166:cytology*
C503924:Pep-1 peptide; D010455:Peptides; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D003543:Cysteamine