Cloning, Expression, And Purification Of Cold Inducible RNA-binding Protein And Its Neuroprotective Mechanism Of Action

BACKGROUND Cold-inducible RNA-binding protein(CIRP) is known to be induced neuroprotection in response to hypothermia. In our preliminary studies, we find it could inhibits H2O2 induced apoptosis in rats neuron. In this study, we report an effective expression and purification approach for this protein, and researched the potential protective effects of rCIRP against neuronal apoptosis.METHODS The cDNA encoding CIRP was expressed by the prokaryotic expression system pGEX-4T-1. We use SP-Sepharose and Sephacryl S-200 columns to purify rCIRP. After cultured in rCIRP for 1 hours, Neuro2a cell was treated with H2O2 (100 μmol/L) for 24 hours. The cells were quantified using the MTT assay and Western blotting were performed for measuring cell cycle signal transduction pathway.RESULTS The survival rates of Neuro2a stimulated by H2O2 decrease significantly compared to the normal conditions. But compared to CIRP group, there is a significantly inhibitive effect with the MTT assay. Western blotting analysis showed that the p-Akt and p-Erk levels stimulated by CIRP were significantly higher than those in the H2O2 group. These results indicate that CIRP induces the activation of transcription factors Akt and Erk in Neuro2a cells.CONCLUSIONS:Our findings suggest that CIRP protein could exerts protective effect without hypothermia, and it probably be a new agent in the neuroprotective field.