Erythropoietin-beta-D-galactosidase. The generation, purification and use of a fusion protein.
A human erythropoietin (Epo) cDNA fragment encoding the complete erythropoietin peptide sequence was fused to the 3'-end of the lacZ gene in the polylinker region of the high expression vector, pUR 278. Escherichia coli bacteria were transformed with the recombinant plasmid harboring the hybrid Epo-beta-D-galactosidase gene. After induction with isopropyl-thiogalactoside large amounts of the fusion protein, Epo-beta-D-galactosidase were synthesized in the transformed bacteria. The fusion protein was partially purified and shown to exhibit intact galactosidase enzymatic activity. Although no biological activity of the Epo counterpart of the fusion protein was detected both in an in vivo and in an in vitro bioassay, the fusion protein served as an effective antigen for the production of anti-erythropoietin antibodies. Antifusion protein antibodies raised in rabbits were shown to react with the intact human Epo molecule from erythropoietin producing culture supernatants. The affinity of these anti-fusion protein antibodies was sufficiently high to permit the development of a sensitive radioimmunoassay for human Epo. This fusion protein approach is a relatively straightforward and rapid method of generating antibodies with specificity for any protein encoded by a cloned eukaryotic gene.
D000818:Animals; D000917:Antibody Formation; D006224:Cricetinae; D004274:DNA, Recombinant; D004921:Erythropoietin / Q000097:blood / Q000235:genetics* / Q000276:immunology; D005696:Galactosidases / Q000235:genetics*; D006801:Humans; D007763:Lac Operon; D011817:Rabbits; D011863:Radioimmunoassay; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification*; D012995:Solubility; D001616:beta-Galactosidase / Q000235:genetics* / Q000276:immunology
D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004921:Erythropoietin; D005696:Galactosidases; D001616:beta-Galactosidase