Active human erythropoietin expressed in insect cells using a baculovirus vector: a role for N-linked oligosaccharide.
Biologically active recombinant human erythropoietin has been expressed at high levels in an insect cell background. Expression involved the preparation of a human erythropoietin cDNA, the transfer of this cDNA to the Autographa californica nuclear polyhedrosis virus (AcNPV) genome under the polyhedrin gene promoter, and the subsequent infection of Spodoptera frugiperda cells with recombinant AcNPV. Erythropoietin cDNA was prepared through the expression of the human erythropoietin gene in COS cells using pSV2 and the construction of a COS cell cDNA library in bacteriophage Lambda GT10. Prior to transfer to the AcNPV genome, erythropoietin cDNA isolated from this library was modified at the 3'-terminus in order to replace genomic erythropoietin for SV40 cDNA derived from pSV2. Transfer of this cDNA to AcNPV and the infection of S. frugiperda cells with cloned recombinant virus led to the secretion of erythropoietin: based on bioassay, rates of hormone secretion (over 40 U/ml per h) were 50-fold greater than observed for COS cells. The purified recombinant product possessed full biological activity (at least 200,000 U/mg), but was of lower Mr (23,000) than human erythropoietin produced in COS cells (30,000) or purified from urine (30,000 to 38,000). This difference was attributed to the glycosylation of erythropoietin in S. frugiperda cells with oligosaccharides of only limited size. Further removal of N-linked oligosaccharides from this Mr 23,000 hormone using N-Glycanase yielded an apo-erythropoietin (Mr 18,000) which possessed substantially reduced biological activity. These results indicate that glycosylation, but not the normal processing of oligosaccharides to complex types, is required for the full hormonal activity of human erythropoietin during red cell development.
D017485:1-Deoxynojirimycin; D000818:Animals; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004921:Erythropoietin / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005822:Genetic Vectors; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007304:Insect Viruses / Q000235:genetics*; D007313:Insecta; D009844:Oligosaccharides / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin; D017485:1-Deoxynojirimycin; D004247:DNA; D005944:Glucosamine