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Transient and stable transfection of Chinese hamster ovary cells with the recombinant feline erythropoietin gene and expression, purification, and biological activity of feline erythropoietin protein

Susan L. BaldwinBiologics Division, Heska Corporation, 1613 Prospect Pkwy, Fort Collins, CO 80525.
Present address is the Department of Immunology, School of Medicine, Health Sciences Center, University of Colorado, Denver, CO 80262.

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Tim D. PowellBiologics Division, Heska Corporation, 1613 Prospect Pkwy, Fort Collins, CO 80525.

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Ramani S. WonderlingBiologics Division, Heska Corporation, 1613 Prospect Pkwy, Fort Collins, CO 80525.
Present address is Diagnostics Division, Abbott Laboratories, 100 Abbott Park Rd, Abbott Park, IL 60064.

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Katherine C. L. KeiserBiologics Division, Heska Corporation, 1613 Prospect Pkwy, Fort Collins, CO 80525.
Present address is Development Division, Pharmaceutical Product Development, 8500 Research Way, Middleton, WI 53562.

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Tony MoralesBiologics Division, Heska Corporation, 1613 Prospect Pkwy, Fort Collins, CO 80525.
Present address is Protein Chemistry Group, Array BioPharma, 3200 Walnut St, Boulder, CO 80301.

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Shirley HunterBiologics Division, Heska Corporation, 1613 Prospect Pkwy, Fort Collins, CO 80525.

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Martin McDermottBiologics Division, Heska Corporation, 1613 Prospect Pkwy, Fort Collins, CO 80525.
Present address is Department of Protein Chemistry, Gilead Sciences, 333 Lakeside Dr, Foster City, CA 94404.

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Steven V. RadeckiBiologics Division, Heska Corporation, 1613 Prospect Pkwy, Fort Collins, CO 80525.

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Michael J. MilhausenBiologics Division, Heska Corporation, 1613 Prospect Pkwy, Fort Collins, CO 80525.

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Abstract

Objective—To use transient and stable transfection of Chinese hamster ovary cells to clone the gene encoding feline erythropoietin (feEPO) protein, characterize the expressed protein, and assess its biological activity.

Sample Population—Cultures of Chinese hamster ovary or TF-1 cells.

Procedure—The gene encoding feEPO was cloned into a eukaryotic expression plasmid. Chinese hamster ovary cells were transiently or stably transfected with the plasmid. Expressed recombinant feEPO (rfeEPO) protein was purified from transiently transfected cells. The protein was characterized by use of SDS gel electrophoresis and western blot analysis. Biological activity was assessed by measuring thymidine incorporation by TF-1 erythroleukemic cells.

Results—Purified rfeEPO from supernatants of transiently transfected cells was determined to be 34 to 40 kilodaltons (kd) by use of SDS gel electrophoresis, whereas the molecular weight predicted from the amino acid sequence was 21.5 kd. The banding pattern and high molecular weight suggested the protein was glycosylated. The rfeEPO proteins derived from transient or stable transfections subsequently were determined to be biologically active in vitro.

Conclusions and Clinical Relevance—The gene encoding feEPO can be transfected into eukaryotic cells, and the expressed rfeEPO protein is biologically active in vitro. Cats with chronic renal failure often are anemic as a result of reduced expression of erythropoietin (EPO). Treatment with human-derived EPO stimulates RBCs in anemic cats; however, treatment is often limited by the development of antibodies directed against the recombinant human protein, which can then cross-react with endogenous feEPO. Recombinant feEPO may prove beneficial for use in cats with chronic renal failure. (Am J Vet Res 2003; 64:1465–1471)

Abstract

Objective—To use transient and stable transfection of Chinese hamster ovary cells to clone the gene encoding feline erythropoietin (feEPO) protein, characterize the expressed protein, and assess its biological activity.

Sample Population—Cultures of Chinese hamster ovary or TF-1 cells.

Procedure—The gene encoding feEPO was cloned into a eukaryotic expression plasmid. Chinese hamster ovary cells were transiently or stably transfected with the plasmid. Expressed recombinant feEPO (rfeEPO) protein was purified from transiently transfected cells. The protein was characterized by use of SDS gel electrophoresis and western blot analysis. Biological activity was assessed by measuring thymidine incorporation by TF-1 erythroleukemic cells.

Results—Purified rfeEPO from supernatants of transiently transfected cells was determined to be 34 to 40 kilodaltons (kd) by use of SDS gel electrophoresis, whereas the molecular weight predicted from the amino acid sequence was 21.5 kd. The banding pattern and high molecular weight suggested the protein was glycosylated. The rfeEPO proteins derived from transient or stable transfections subsequently were determined to be biologically active in vitro.

Conclusions and Clinical Relevance—The gene encoding feEPO can be transfected into eukaryotic cells, and the expressed rfeEPO protein is biologically active in vitro. Cats with chronic renal failure often are anemic as a result of reduced expression of erythropoietin (EPO). Treatment with human-derived EPO stimulates RBCs in anemic cats; however, treatment is often limited by the development of antibodies directed against the recombinant human protein, which can then cross-react with endogenous feEPO. Recombinant feEPO may prove beneficial for use in cats with chronic renal failure. (Am J Vet Res 2003; 64:1465–1471)