[Expression and purification of PEP-1-EGFP fusion protein and its transduction into human umbilical vein endothelial cells].
OBJECTIVE
To construct the expression vector pET15b-pep-1-EGFP and purify the fusion protein PEP-1-EGFP expressed in E. coli BL21(DE(3)) for evaluating the cell-penetrating capability of the cell-penetrating peptide PEP-1.


METHODS
Two oligonucleotides encoding PEP-1 was synthesized and annealed to generate PEP-1-encoding DNA. The recombinant plasmid pET15b-pep-1-EGFP was constructed by inserting PEP-1-encoding DNA and enhanced green fluorescent protein (EGFP) cDNA into pET15b. The fusion protein PEP-1-EGFP expressed in E. coli BL21(DE(3)) was purified with Ni(2+)-resin affinity chromatography and transduced into human umbilical vein endothelial cells.


RESULTS
Sequence analysis confirmed successful construction of the expression vector pET15b-pep-1-EGFP, and the fusion protein PEP-1-EGFP was expressed and purified efficiently with a yield of approximately 14.15 mg/100 ml bacteria medium. SDS-PAGE and Western blotting identified the purified protein as PEP-1-EGFP, and the cell-penetration assay verified that the fusion protein could be transduced into human umbilical vein endothelial cells.


CONCLUSIONS
The successful expression and purification of PEP-1-EGFP and its efficient transduction into human umbilical vein endothelial cells provides a basis for PEP-1-mediated biomacromolecular transduction in protein therapy.
Dong
Xiao
X
Institute of Clinical Medicine, People's Hospital of Yunyang Medical College, Shiyan 442000, China. sydx7226@sina.com
Wang
Jia-ning
JN
Huang
Yong-zhang
YZ
Guo
Ling-yun
LY
D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D003543:Cysteamine / Q000031:analogs & derivatives* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; 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