Biology Reference
In-Depth Information
change in the number of circulating red blood cells. Production of new red
blood cell slows until the sensor cells recognize a need for increased erythro-
poiesis. Some EPO, albeit in trace amounts, is always detectable in the circu-
lating blood, even in patients with total kidney failure, suggesting that a sub-
set of cells provides a continuous output of EPO even when oxygen delivery
is normal.
Isolation of EPO from urine
Even though the amounts of endogenous EPO increase under conditions of
hypoxia or anemia, EPO represents a minor fraction of the total protein in the
blood. A significant obstacle to the development of EPO as a therapeutic agent
was the difficulty in isolating and purifying adequate amounts of the hormone
to allow for its characterization. Several groups attempted to purify human [31,
32] and sheep [33, 34] EPO. The results were inconclusive, with the purity of
the product questionable or produced in insufficient amounts to allow chemi-
cal characterization.
In 1977, Miyake, Kung,and Goldwasser succeeded in isolating and purify-
ing milligram amounts of EPO from 1500 L of urine from patients with aplas-
tic anemia [35]. Patients with aplastic anemia characteristically overproduce
EPO, however the amounts present are still small. Large volumes of urine were
essential for the recovery of a sufficient amount to purify EPO.
Miyake et al. [35], using a seven-step process that included ion exchange
chromatography, ethanol precipitation, gel filtration, and adsorption chro-
matography, produced a preparation with a potency of 70,400 units/mg pro-
tein, 21% yield, and a purification factor of 930. This method allowed the pro-
duction of enough material to partially characterize the hormone. With a
source of EPO now available, a strategy for the cloning and expression of the
human gene was devised.
Cloning of the
EPO
gene
In the 1980s, the nucleic acid sequences for cynomalogous monkey and human
EPO were finally isolated and characterized, each by a different method.
Several hurdles needed to be overcome, including the limited amount of data
about the primary structure of human or monkey EPO, lack of a known source
of mRNA, no information about the genomic structure of the gene, lack of
simple tests to confirm that the cloned sequence encoded the
EPO
gene, and
controversy about the induction mechanism for EPO production due to hypox-
ia. The difficulty in cloning the gene was further complicated by an inability
to determine which step in the process was responsible for the failure.
In 1981, Goldwasser presented the first 26 amino acids of the protein at a
meeting. Sue and Sytkowski [36] published the sequence in a paper that
