An erythropoietin fusion protein comprised of identical repeating domains exhibits enhanced biological properties.
The hematopoietic growth factor erythropoietin (Epo) initiates its intracellular signaling cascade by binding to and inducing the homodimerization of two identical receptor molecules. We have now constructed and expressed in COS cells a cDNA encoding a fusion protein consisting of two complete human Epo domains linked in tandem by a 17-amino acid flexible peptide. On SDS-polyacrylamide gel electrophoresis, the Epo-Epo fusion protein migrated as a broad band with an average apparent molecular mass of 76 kDa, slightly more than twice the average apparent molecular mass of Epo, 37 kDa. Enzymatic N-deglycosylation resulted in an Epo-Epo species that migrated on SDS-polyacrylamide gel electrophoresis as a narrow band with an average apparent molecular mass of 39 kDa. The specific activity of the Epo-Epo fusion protein in vitro (1,007 IU/microgram; 76 IU/pmol) was significantly greater than that of Epo (352 IU/microgram; 13 IU/pmol). Moreover, secretion of Epo-Epo by COS cells was 8-fold greater than that of Epo. Subcutaneous administration of a single dose of Epo-Epo to mice resulted in a significant increase in red blood cell production within 7 days. In contrast, administration of an equivalent dose of conventional recombinant Epo was without effect. The pharmacokinetic behavior of Epo-Epo differed significantly from that of Epo. The results suggest that Epo-Epo may have important biological and therapeutic advantages.
Sytkowski
A J
AJ
Laboratory for Cell and Molecular Biology, Division of Hematology, Beth Israel Deaconess Medical Center, Massachusetts 02215, USA. asytkows@caregroup.harvard.edu
Lunn
E D
ED
Risinger
M A
MA
Davis
K L
KL
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000235:genetics* / Q000493:pharmacokinetics / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D007279:Injections, Subcutaneous; D051379:Mice; D008970:Molecular Weight; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000493:pharmacokinetics / Q000494:pharmacology; D020080:Tandem Repeat Sequences / Q000235:genetics*
D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase