Genetically Fused Protein A-Luciferase for Immunological Blotting Analyses

The gene expression plasmid pMALU5 for the fusion protein of protein A (SpA) with a complete sequence of firefly luciferase (Luc) was constructed. The fused gene was expressed in Escherichia coli, and the resulting SpA–Luc fusion protein was purified by one-step affinity chromatography on IgG–Sepharose. The protein retained both activities: IgG binding capability of protein A and enzymatic activity of luciferase. Blotting analyses were performed with the fusion protein to determine a tumor marker of α-fetoprotein (AFP). AFP was detected at the lowest detection limit of 5 pg by dot blotting and Western blotting. The SpA–Luc fusion protein provides a highly selective, sensitive, and versatile marker for blotting analyses.