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 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.
Zhang
X M
XM
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4529 Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan.
Kobatake
E
E
Kobayashi
K
K
Yanagida
Y
Y
Aizawa
M
M
D015153:Blotting, Western; D002846:Chromatography, Affinity; D004306:Dose-Response Relationship, Immunologic; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D007074:Immunoglobulin G / Q000737:chemistry; D007700:Kinetics; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D012685:Sepharose / Q000737:chemistry; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000509:alpha-Fetoproteins / Q000378:metabolism
D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D000509:alpha-Fetoproteins; D012685:Sepharose; D008156:Luciferases