Faculty Bibliography

Human erythropoietin (Ep) cDNA has been cloned in Escherichia coli by using pBR322 as a vector. Polyadenylylated RNA was isolated from selected human renal carcinomas with elevated Ep titers. The presence of Ep mRNA was detected by immunoprecipitation of in vitro translation products with monoclonal antibody to human Ep. Double-stranded cDNA was synthesized and inserted into the Pst I site of pBR322 by homopolymeric dG . dC tailing. The cDNA library was initially screened by colony hybridization with 32P-labeled cDNA synthesized from size-fractionated mRNA enriched in Ep message. Positive colonies were further screened immunologically by in situ radioimmunoassay with monoclonal antibody to human Ep. Three positive clones were identified that express the Ep gene sequences as a beta-lactamase fusion protein. These clones contain inserts of approximately 1400, 600, and 200 base pairs. Human renal Ep mRNA, of which the translation products immunoreact with anti-Ep on immunoblots, was hybrid-selected by plasmid DNA from these recombinants. Purified human Ep competes with 35S-labeled hybrid-selected translation products for antibody binding

A new preparative method for isolation of human urinary erythropoietin (Ep) has been developed using hydrophobic interaction chromatography on Phenyl-Sepharose CL4B. Crude urine and urine concentrates from anemic patients were used directly without prior manipulation. In addition to facilitating hydrophobic interactions, Phenyl-Sepharose provided pi-pi interactions between its phenyl group on the gel matrix and the aromatic amino acid residues of Ep, and thus contributed to specific resolution. Over 90% of the urinary contaminants were excluded from the column, and Ep was selectively bound. Its activity was eluted with 20% ethylene glycol in 10 mM NaOH containing 4 M guanidine hydrochloride. This single step offered a mean purification factor of 110 with a recovery of 85%