Production and characterization of recombinant equine prorelaxin.
Relaxin is a peptide hormone produced by a wide variety of mammals. In the horse, the placenta is the major source of relaxin. Since pure equine relaxin is difficult to obtain to study its role in the pregnant mare, the objectives of this study were to produce recombinant equine prorelaxin and characterize its immunological and biological activity. First, an equine relaxin gene cassette was transfected into immortalized bovine mammary epithelial (MAC-T) cells. Second, immunological activity of media conditioned by transfected MAC-T cells was tested by Western blotting and quantified using a homologous equine radioimmunoassay. Finally, bioactivity of the conditioned media was tested using the human monocyte cell line, THP-1, which exhibits a rapid and dose-dependent increase in the accumulation of cAMP upon binding relaxin. The results showed that conditioned media, concentrated 5x, yielded 4.11 +/- 0.81 ng/ml recombinant equine prorelaxin. In addition, a 19 kDa immunoreactive band, corresponding to the expected size of equine prorelaxin, was visualized by SDS-PAGE. THP-1 cells incubated with conditioned media (5x) from transfected cells, in the presence of forskolin (1 microM) and isobutylmethylxanthine (50 microM), showed an increase in cAMP production over media from mock-transfected cells alone. In conclusion, recombinant equine prorelaxin secreted by MAC-T cells was both immunologically and biologically active. This study demonstrates the first attempt to produce recombinant equine prorelaxin, important for further study of the role of relaxin in the mare.
Neumann
Jennifer L
JL
Department of Animal Sciences, Rutgers University, 84 Lipman Drive, New Brunswick, NJ 08901, USA.
Lazaris
Anthoula
A
Huang
Yue-Jin
YJ
Karatzas
Costas
C
Ryan
Peter L
PL
Bagnell
Carol A
CA
D000818:Animals; D001681:Biological Assay / Q000662:veterinary; D015153:Blotting, Western / Q000662:veterinary; D002417:Cattle; D002460:Cell Line; D017077:Culture Media, Conditioned; D000242:Cyclic AMP / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics; D005260:Female; D006736:Horses / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D016254:Mutagenesis, Insertional; D011498:Protein Precursors / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D012065:Relaxin / Q000096:biosynthesis* / Q000235:genetics* / Q000494:pharmacology / Q000502:physiology; D014162:Transfection
D017077:Culture Media, Conditioned; D011498:Protein Precursors; D011994:Recombinant Proteins; C038656:prorelaxin; D012065:Relaxin; D004247:DNA; D000242:Cyclic AMP