COS-1 CELL EXPRESSION AND ONE-STEP AFFINITY PROTEIN-PURIFICATION AND ACTIVITY OF EPITOPE-TAGGED HUMAN ERYTHROPOIETIN AND OF SITE-DIRECTED MUTANTS

Recombinant human erythropoietin (rhEPO) is an important glycoprotein hormone which has been successfully used in the treatment of anaemia. To facilitate the rapid evaluation of wild-type and mutant forms of rh EPO in structure-function studies, we have developed an expression sys tem in which the recombinant hormone is tagged at the C-terminus with a c-myc peptide, One-step affinity purification of culture supernatant s on an anti-myc antibody column yielded proteins which were greater t han 50% pure with a specific activity of 300000 U/mg, in agreement wit h the value of wild-type protein. We conclude that the additional myc- peptide does not affect receptor binding. The expression system was us ed to study three mutants in which the N-glycosylation sites were chan ged to cysteines (Asn24Cys, Asn38Cys and Asn83Cys). Specific activitie s of these cysteine mutants were significant, but reduced (60%, 22% an d 70%, respectively), compared to wild-type. The reduction in specific activity may be due to reduced stability of the mutant proteins.