High-level expression and purification of Tat-haFGF 19-154

Human acidic fibroblast growth factor (haFGF) stimulates repair and regeneration of central and peripheral nerves after various injuries. However, it is unable to cross the blood–brain barrier (BBB). To produce a therapeutic haFGF with cell-permeable activity, we fused the haFGF 19-154 gene with Tat-PTD. After its construction by a single-step insertion of a polymerase chain reaction (PCR)–amplified coding sequence, the vector pTat-haFGF 19-154 -His was expressed in Escherichia coli BL21 (DE3) cells. The optimal expression level of the soluble fusion protein was up to 36.7% of the total cellular protein. The recombinant Tat-haFGF 19-154 -His was purified by a combination of Ni–NTA affinity, Sephadex G-25, and heparin affinity chromatography to 95% as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The final yield was 171 mg/l culture. Purified Tat-haFGF 19-154 -His had distinct mitogenic activity in Balb/c 3T3 cells, as measured by methylthiazoletetrazolium (MTT) assay and its ED 50 was 3.931 × 10 −4 µmol/l. Tat-haFGF 19-154 -His protein intravenously injected at the dose of 10 mg/kg could be detected in the pallium and hippocampi.