title: Recombinant fusion proteins of protein A and protein G with glutathione S-transferase as reporter molecules.
abstract: The regions encoding the IgG-binding domains of protein A (PA) and protein G (PG) were cloned into the bacterial expression vector pGEX. Both proteins were expressed in Escherichia coli as fusion proteins with glutathione S-transferase (PA-GST and PG-GST) and were found to be soluble, abundant and easily purified in one step from the bacterial lysate by affinity chromatography on immobilized glutathione. Yields of 50 mg/litre of cultures were obtained. Both purified fusion proteins were shown to be functional in a variety of immunochemical procedures. In radial diffusion tests, PA-GST precipitated IgG from human, squirrel monkey, rabbit, dog, cat and pig but not mouse, sheep, goat, cow, horse or chicken. PG-GST formed precipitin bands with IgG from human, rabbit, mouse, pig, sheep, goat, cow and horse but not squirrel monkey, dog, cat and chicken IgG. The fusion proteins were shown to function as effective detection reagents in ELISA and Western blotting. Glutathione agarose beads with bound fusion protein were shown to be useful for immunoprecipitation.
authors.affiliation: Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia.
authors.forename: A M
authors.initials: AM
authors.lastname: Lew
authors.forename: D J
authors.initials: DJ
authors.lastname: Beck
authors.forename: L M
authors.initials: LM
authors.lastname: Thomas
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005982:Glutathione Transferase; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000276:immunology; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology; D013205:Staphylococcal Protein A / Q000096:biosynthesis* / Q000276:immunology; D013379:Substrate Specificity
chemical_list: C029152:G-substrate; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D005982:Glutathione Transferase