Objective: To construct a eukaryotic expression vector for His-tagged human soluble receptor for advanced glycation end products (hsRAGE), and study the function and biological activity of the fusion protein.
Methods: The coding sequence of hsRAGE was amplified from the recombinant pCI-neo/RAGE by PCR method. The fragment was cloned into pET-14b vector and identified by digestion with restriction enzymes and sequence analysis. After transformation of the vector into E. coli BL21(DE3), the fusion protein was expressed successfully in response to IPTG induction in the form of inclusion body. After extraction from the bactrerial cells and washing, the fusion protein was dissolved in 8 mol/L urea and purified using immobilized metal ion affinity chromatography. Renaturing of the fusion protein occurred after gradually removal of the denaturants by dialysis. The protein was subsequently identified by Western blotting, enzyme-linked immunosorbent assay (ELISA) and blockade experiments in the endothelial cells.
Results: The recombinant hsRAGE/pET-14b was verified by enzyme digestion and sequencing. A fusion protein with a molecular weight of about 45 kD was purified, which could interact with anti-RAGE antibody and AGE and block AGE-induced nuclear factor (NF)-kB activation in the endothelial cells.
Conclusions: His-hsRAGE fusion protein has been cloned, expressed and purified successfully and identified to possess immunogenicity, binding activity to AGE and blocking effect on AGE-RAGE.