Genetically fused protein A-luciferase for immunological blotting analyses |
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Authors: | Zhang X M Kobatake E Kobayashi K Yanagida Y Aizawa M |
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Institution: | Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4529 Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan. |
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Abstract: | 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 alpha-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. |
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