Abstract
An improved method for the purification of human erythropoietin with high in vivo activity from urine was developed. This method involved ion-exchange, gel permeation, affinity chromatography, and reverse-phase chromatography but did not involve any stabilizing procedures. The purified human urinary erythropoietin showed a single broad band with a molecular weight between 37000 and 39000 Da on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and had an in vivo specific activity of 160000 IU/mg comparable to that of human erythropoietin produced in recombinant Chinese hamster ovary cells. We found that omission of the phenol treatment and ethanol precipitation which are usually used in the purification of human urinary erythropoietin greatly improved the biological activity of the final product. Phenol treatment followed by ethanol precipitation did not affect the amino acid composition but decreased the apparent molecular weight and N-acetylglucosamine content of human urinary erythropoietin. These findings suggest that phenol treatment followed by ethanol precipitation does not restore erythropoietin with high branched sugar chains which would have high in vivo specific activity as reported previously (M. Takeuchi, et al. (1989) Proc. Natl. Acad. Sci. U.S.A., 86, 7819-7822).
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