Abstract: Objective To construct the expression vector pET15b-pep-1-EGFP and purify the fusion protein PEP-1-EGFP expressed in E.coli BL21(DE3) 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(DE3) was purified with Ni2+-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. Conclusion 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.