Detection of erythropoietin and erythropoietin receptor

Materials and Methods

[0052]Glutathione (GSH)-agarose, pGEX-2T expression vector and Sephadex G-50 were purchased from Pharmacia (Mechanicsburg, Pa.). PCR reagents were from Perkin-Elmer Cetus (Norwalk, Conn.) and Affigel 15 was from BioRad (Richmond, Calif.). Bacteriophage T4 DNA ligase, restriction enzymes and isopropylthio-.beta.-D-galactoside (IPTG) were purchased from BRL Gibco (Gaithersburg, Md.). Geneclean II was from Bio 101, La Jolla, Calif. Nitrocellulose was from Schleicher & Schuell Co. (Keene, N.H.). Chemiluminescence (ECL) reagents and .sup.125 I-Epo were from Amersham (Arlington Heights, Ill.) and unlabeled Epo was a gift of Chugai-Upjohn (Rosemont, Ill.). Phenylmethylsulfonylfluoride (PMSF), diisopropylfluorophosphate (DFP), thrombin, trypsin and Triton X-100, were from Sigma Chemical Company (St. Louis, Mo.). Biotinylated rabbit anti-sheep antibodies and avidin-horseradish peroxidase were from Pierce Co. (Rockford, Ill.). LAP37, a full-length human erythropoietin recept

Construction of EpoR cDNA Recombinant Vector

[0053]A recombinant plasmid expression vector, pJYL26, was constructed from a PCR product having the human Epo receptor extracellular domain coding sequence and from the plasmid vector pGEX-2T. The construction of this plasmid is explained below.

[0054]PCR amplification was carried out using a full-length human EpoR cDNA, LAP37, as a template. The 5′-sense primer was 5′-TTGGATCCGCGCCCCCGCCTAAC-3′ (SEQ ID NO: 1). This primer has a BamH1 linker sequence at the 5′ end, followed by the coding sequence for amino acids 25 through 29 of the full length human EpoR protein. The 3′-antisense primer was 5′-TGAATTCGGGGTCCAGGTCGCT-3′ (SEQ ID NO: 2). This primer has an EcoR1 linker followed by sequence complementary to the coding sequence for amino acids 226 through 222 of full length EpoR. Using a Perkin Elmer-Cetus PCR kit, PCR was carried out with 0.1 μg of LAP37 cDNA, 20 pM of each primer, 1.25 mM dNTP mixture (dGTP, dCTP, dTTP and dATP), 0.5 μl of Taq

Purification of EpoRex-th Fusion Protein

[0059]This example teaches the production and purification of a fusion protein having two segments. The first segment is a polypeptide, GST, with a thrombin cleavage site at the carboxyl terminus. The second segment, fused to the first segment at the thrombin cleavage site, is the extracellular domain of human Epo receptor. The fusion protein EpoRex-th, containing GST and Epo-bp, is purified by GSH-agarose affinity chromatography.

[0060]Transformed E. coli containing the recombinant vector pJYL26 were grown overnight at 37° C. with vigorous shaking in 400 ml of LB medium with 100 μg / ml of ampicillin. The following day, the culture was diluted in 4 liters of fresh LB / Amp media and incubated for another 90 min before adding 1 mM isopropylthio-β-D-galactoside (IPTG). After 4 hours of IPTG induction, the cells were pelleted at 3,000×g at 4° C. for 15 min and resuspended in 160 ml of lysis buffer, containing 50 mM sodium phosphate, pH 7.4, 10 mM β-