OBJECTIVETo construct prokaryotic expression vector of pET15b-PEP-1-SOD1 and investigate whether PEP-1-SOD1 fusion protein could be transduced into human umbilical vein endothelial cells (HUVECs) and the effects on hypoxia/reoxygenation injury.

METHODSThe recombinant plasmids pET15b-SOD1 and pET15b-PEP-1-SOD1 were constructed and transformed into E. coli BL21 (DE3) to express SOD1 and PEP-1-SOD1 with an N-terminal His-tag. The purified SOD1 and PEP-1-SOD1 were incubated with HUVECs and the viability (MTT assay) and the release of lactate dehydrogenase (LDH) in culture medium were determined in the hypoxia/reoxygenation injury model. The morphological changes were observed under an inverted phase contrast microscope. The content of malondialdehyde (MDA) in HUVECs was also determined with the method of thiobarbituric acid.

RESULTSPEP-1-SOD1 fusion protein could be transduced into cultured HUVECs in a time- and dose-dependent manner. The intracellular enzymatic activity of PEP-1-SOD1 after 30 min incubation with HUVECs was significantly higher than control group (60.88 U/ml +/- 6.73 U/ml vs. 41.06 U/ml +/- 4.19 U/ml, P < 0.01). The transduced PEP-1-SOD1 protein was enzymatically stable for 24 h within cells. After hypoxia/reoxygenation injury, control HUVECs shrunk, became round-shaped and intercellular space increased, while these morphological changes were not observed in PEP-1-SOD1 transduced HUVECs. PEP-1-SOD1 transduction also markedly increased the viability, decreased LDH leakage into culture media and reduced the content of MDA post hypoxia/reoxygenation.

CONCLUSIONSPEP-1-SOD1 fusion protein could be efficiently transduced into HUVECs in a natively active form, and the delivered enzymatically active PEP-1-SOD1 exhibits cellular protection against hypoxia/reoxygenation injury in HUVECs. The transduction of SOD1 mediated by cell-penetrating peptide, PEP-1, provides a basis for further research on the prevention of ischemia/reperfusion injury in vivo.