Abstract
Thermal stress was used to assess the stability of recombinant human erythropoietin (EPO) derived from Chinese hamster ovary cells. In 20 mM phosphate at pH 7.0, this protein had a highly reversible thermal unfolding as observed by far UV circular dichroism (CD) and native gel analysis, with no indication of protein aggregation. It had a relatively low melting temperature at 53°C. Assuming a two-state transition, the observed reversibility permits thermodynamic analysis of the unfolding of EPO, which shows that the free energy of unfolding at 25°C is only 6-7 kcal/mol. Upon heating to 79°C over 30 min, however, this protein does undergo aggregation as assessed by native gel. In 20 mM phosphate and citrate at pH 7.0, the results are similar, i.e., EPO suffered a substantial aggregation, while it showed little aggregation in 20 mM Tris or histidine at pH 7.0 and 20 mM glycine at pH 6.3 under identical heat treatment.