Purification of human erythropoietin to homogeneity by a rapid five- step
procedure
G Krystal, HR Pankratz, NM Farber and JE Smart
Human urinary erythropoietin (Ep) has been purified using a simple five-
step procedure to yield preparations with potencies of 80,000 U/mg in 25%
yield. The five steps involve: (1) affinity chromatography on CM Affi-Gel
Blue, (2) chromatofocusing, (3) wheat germ lectin (or hydroxylapatite)
chromatography, (4) reverse-phase high-performance liquid chromatography
(HPLC) using a phenyl column, and (5) preparative sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Ep activity was
determined at each stage using a highly sensitive and specific in vitro
assay that measures [3H]-thymidine incorporation into erythroid cells from
spleens of phenylhydrazine-treated mice. The step 5 material was also
tested with the in vivo polycythemic mouse assay procedure and was found to
have a similar potency to that obtained in the [3H]-thymidine in vitro
assay. SDS-PAGE analysis of the step 5 material revealed a single 38.5-kd
band that comigrated with Ep bioactivity. Homogeneity was confirmed by
amino acid sequence analysis. Starting with urine containing approximately
13 U/mg of protein, the cumulative degrees of purification achieved with
each step were: step 1,25-fold; step 2, 75-fold; step 3, 300-fold; step 4,
1,500-fold; and step 5, 5,000-fold. Corresponding overall recoveries after
each step were: greater than 100%, 70%, 45%, 30%, and 25%. These recoveries
could be obtained when as little as 5,000 U of starting urinary Ep were
processed because of the introduction of Tween 20 and SDS into buffers used
at various stages of the purification procedure. In addition, a rapid
method for determining Ep purity which involves reverse-phase HPLC of
trypsinized 125I-labeled Ep is presented. This allows the establishment of
purity with far less material than is required for amino acid sequencing.
Volume 67,
Issue 1,
pp. 71-79,
01/01/1986
Copyright © 1986 by The American Society of Hematology
