Biology Reference
In-Depth Information
cleotides and a peptide sequence derived from urinary EPO supplied by
Miyake. Cloning of additional
EPO
genes from other species used the
sequence information from the human and monkey genes and proceeded rap-
idly. The mouse
EPO
gene was cloned using monkey and human EPO DNA
segments as hybridization probes [42, 43]. Subsequently,
EPO
genes from
other species were cloned by hybridization or by polymerase chain reaction
using probes or primers based on the known EPO sequences, including the
genes from the rat [44], pig [45, 46], sheep [45, 47], and cow [48], among
other animals.
Expression of the
EPO
gene
Clones of the gene for EPO were inserted into CHO cells, which synthesized
the 193 amino acid precursor protein, removed the signal peptide and carboxy-
terminal arginine, added
N
- and
N O
-linked carbohydrate to glycosylation sites,
and released the mature protein into the culture medium. Immunologic, bio-
logic, and biochemical assays showed that the recombinant hormone had the
in vivo
biologic activity and was immunologically equivalent to human EPO,
as revealed within the limits of the available assays [39]. EPO expressed by
CHO cells has a molecular weight of 30.4 Kd and contains 40% carbohydrate
[49].
Discussion
The isolation and expression of the gene for human EPO was a major
achievement that capped almost 155 years of exploration into the nature of
anemia and the production of red blood cells. EPO is present in minute quan-
tities in the blood,and difficulty in isolating and purifying the hormone in
amounts that would allow investigation of its properties posed a significant
obstacle to the development of EPO as a therapeutic agent. After Miyake,
Kung, and Goldwasser developed a technique to purify urinary EPO, a strate-
gy for the cloning and expression of the human gene could be devised. The
innovative approach of Lin et al. allowed the successful isolation, cloning,
sequencing, and development of the recombinant protein. Large-scale produc-
tion began and clinical trials started. The recombinant protein, epoetin alfa,
produced dose-dependent increases in erythropoiesis that paralleled the
expected response to endogenous EPO. The commercial production of epoet-
in alfa has been translated into benefits for millions of patients (Tab.1).
Epoetin alfa ameliorates the debilitating symptoms of anemia and allows these
patients to have more normal lives. Other chapters in this volume will elabo-
rate on the clinical use, production, formulation, and other important and
evolving aspects in the study of rHuEPO.
