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.
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.


METHODS
Cultures of Chinese hamster ovary or TF-1 cells.


METHODS
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
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.
Baldwin
Susan L
SL
Biologics Division, Heska Corporation, 1613 Prospect Pkwy, Fort Collins, CO 80525, USA.
Powell
Tim D
TD
Wonderling
Ramani S
RS
Keiser
Katherine C L
KC
Morales
Tony
T
Hunter
Shirley
S
McDermott
Martin
M
Radecki
Steven V
SV
Milhausen
Michael J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002415:Cats; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004921:Erythropoietin / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism*; D015870:Gene Expression; D008969:Molecular Sequence Data; D009281:Naphthalenes; D010082:Oxepins; D011994:Recombinant Proteins; D013936:Thymidine / Q000378:metabolism; D014162:Transfection
C461941:1-phenyl-1,4-epoxy-1H,4H-naphtho(1,8-de)(1,2)dioxepin; D009281:Naphthalenes; D010082:Oxepins; D011994:Recombinant Proteins; D004921:Erythropoietin; D013936:Thymidine