Journal
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY
Volume 65, Issue 6, Pages 1321-1327Publisher
TAYLOR & FRANCIS LTD
DOI: 10.1271/bbb.65.1321
Keywords
erythropoietin; thermal unfolding; aggregation; reversibility; native gel
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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 degreesC. 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 degreesC is only 6-7 kcal/mol, Upon heating to 79 degreesC 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.
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