Cloning and expression of the human erythropoietin gene.

Abstract

The human erythropoietin gene has been isolated from a genomric phage library by using mixed 20-mer and 17-mer oligonucleotide probes. Construction of Oligonucleotide Probes. Purified human urinary Epo'isolated from the urine of patients with aplastic anemia (16) was subjected to tryptic digestion. The resulting fragments were isolated and sequenced by using an Applied Biosystems gas-phase microsequencer (unpublished data). A hexapeptide and a heptapeptide containing the least codon degeneracy were selected for oligodeoxyribonucleotide probe synthesis. The phosphoramidite method was l4sed for oligonucleotide synthesis (19,20). Each' probe mixture contained a pool of 128-oligonucleotide sequences. The probe mixtures were Probe mixture EpV = Val-Asn-Phe-Tyr-Ala-Trp-Lys 3' CAA TTG AAG ATG CGA ACC TT 5'Probe mixture EpQ= Gln-Pro-Trp-Glu-Pro-Leu 3' GTT GGA ACC CTT GGA GA 5'The probe mixtures were labeled at the 5' end with [-32P]ATP, 7500-8000 Ci/mmol (ICN) (1 Ci = 37 GBq), by using T4 polynucleotide kinase (21). Hybridization Procedures. Phage plaques were amplified according to the procedures of Woo (22) except that GeneScreenPlus filters and NZYAM plates [NaCl, 5 g; MgCl2-6H2O, 2 g; NZ-Amine A, 10 g; yeast extract, 5 g; Casamino acids, 2 g; maltose, 2 g; and agar, 15 g (per liter)] were utilized. Phage particles were disrupted and the DNAs were fixed on filters (50,000 plaques per 8.4 x 8.4 cm filter). The air-dried filters were baked at 80'C for 1 hr and then subjected to proteinase K digestion [50 ,ug ofproteinase 'K per ml of buffer solution containing 0.1 M Tris HCl (pH 8.0), 0.15 M NaCl, 10 mM EDTA, and 0.2% NaDodSO4] for 30 min at 550C. Prehybridization with a 1 M NaCl/1% NaDodSO4 solution was carried out at 550C for 4 hr or longer.The hybridization buffer contained 0.025 pmol/ml of each of the 128 probe sequences in 0.9 M NaCl/5 mM EDTA/50 mM sodium phosphate, pH 6.5/0.5% NaDodSO4/100 jg of yeast tRNA per ml. Hybridization was carried out at 480C'for 20 hr by using the EpV probe mixture. This is 2TC below the lowest calculated dissociation temperature (td) (23) for members of the mixture. At the completion of hybridization, the filters were washed three times with 0.9 M NaCl/90 mM sodium citrate, pH 7.0/0. 1% NaDodSO4 at room temperature Abbreviations: Epo, erythropoietin; CHO, Chinese hamster ovary; DHFR, dihydrofolate reductase; kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); SV40, simian virus 40; td, dissociation temperature. 7580The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Nonviral gene delivery of erythropoietin by mesenchymal stromal cells

“…Cloning of the EPO gene in 1985 1 paved the way for the production of recombinant EPO, which has revolutionized the treatment of anemia. Endogenous human EPO is a 165 amino-acid glycoprotein hormone, which is primarily produced by interstitial fibroblasts in the kidneys in response to hypoxemia.…”

Erythropoiesis-Stimulating Agents in Renal Medicine

“…The cloning of the gene encoding human erythropoietin (EPO) in 1985 [2] led to the synthesis of epoetin alfa [3,4] and, subsequently, epoetin beta. Both are synthesized in Chinese hamster ovary (CHO) cells and have the same amino acid sequence as endogenous EPO, but differences in their manufacturing processes lead to small differences in their carbohydrate moieties [5].…”

Recombinant human erythropoietin (rHuEPO): Cross‐linking with disuccinimidyl esters and identification of the interfacing domains in EPO

“…The amino acid sequence of EPO was determined initially by cDNA sequence analysis (Jacobs et al, 1985;Lin et al, 1985) and later confirmed by protein sequence analysis (Lai et al, 1986). It contains both N-linked and O-linked carbohydrates that have been structurally determined (Sasaki et al, 1988).…”