Production and characterization of recombinant equine prorelaxin.
Abstract: 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.
Publication Date: 2005-10-21 PubMed ID: 16274952DOI: 10.1016/j.domaniend.2005.10.001Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research article details the successful production and characterization of recombinant equine prorelaxin, a peptide hormone, vital for studying its role in pregnant horses.
Introduction and Objectives
- The study focuses on relaxin, a peptide hormone produced widely across many mammalian species.
- In horses, the placenta is the major source of relaxin. The problem arises as pure equine relaxin is challenging to obtain in order to study its function in pregnant mares.
- Hence, the main objective of this study is to produce the recombinant form of equine prorelaxin and characterize its immunological and biological activity.
Methodology
- Initially, an equine relaxin gene cassette was inserted into immortalized bovine mammary epithelial (MAC-T) cells.
- Secondly, the immunological activity of media that had been conditioned by the transfected MAC-T cells was investigated using Western blotting techniques and assessed by a homologous equine radioimmunoassay.
- Final stage includes the bioactivity testing. For this, the human monocyte cell line (THP-1) was used. THP-1 cells reveal a rapid and dose-dependent increase in cAMP accumulation upon binding to relaxin hormone.
Results
- Results confirm that the conditioned media, after being concentrated to 5 times the original volume, yielded approximately 4.11 +/- 0.81 ng/ml of recombinant equine prorelaxin.
- Further, a 19 kDa immunoreactive band, symbolizing the estimated size of the equine prorelaxin, was visible during SDS-PAGE (a commonly used method for protein separation and analysis).
- Moreover, THP-1 cells incubated with the conditioned media showed an increase in cAMP production over media from mock-transfected cells alone when exposed to forskolin and isobutylmethylxanthine.
Conclusion
- The study concludes that the recombinant equine prorelaxin secreted by MAC-T cells was both immunologically and biologically active.
- This represents the first successful attempt to produce and characterize recombinant equine prorelaxin, opening avenues for more detailed investigation into the role of relaxin in pregnant mares.
Cite This Article
APA
Neumann JL, Lazaris A, Huang YJ, Karatzas C, Ryan PL, Bagnell CA.
(2005).
Production and characterization of recombinant equine prorelaxin.
Domest Anim Endocrinol, 31(2), 173-185.
https://doi.org/10.1016/j.domaniend.2005.10.001 Publication
Researcher Affiliations
- Department of Animal Sciences, Rutgers University, 84 Lipman Drive, New Brunswick, NJ 08901, USA.
MeSH Terms
- Animals
- Biological Assay / veterinary
- Blotting, Western / veterinary
- Cattle
- Cell Line
- Culture Media, Conditioned
- Cyclic AMP / metabolism
- DNA / chemistry
- DNA / genetics
- Female
- Horses / genetics
- Horses / metabolism
- Humans
- Mutagenesis, Insertional
- Protein Precursors / genetics
- Protein Precursors / physiology
- Recombinant Proteins / biosynthesis
- Recombinant Proteins / genetics
- Recombinant Proteins / pharmacology
- Relaxin / biosynthesis
- Relaxin / genetics
- Relaxin / pharmacology
- Relaxin / physiology
- Transfection
Citations
This article has been cited 2 times.- Cimini D, Corte KD, Finamore R, Andreozzi L, Stellavato A, Pirozzi AV, Ferrara F, Formisano R, De Rosa M, Chino M, Lista L, Lombardi A, Pavone V, Schiraldi C. Production of human pro-relaxin H2 in the yeast Pichia pastoris. BMC Biotechnol 2017 Jan 14;17(1):4.
- Kwok SC, Chakraborty D, Soares MJ, Dai G. Relative expression of proprotein convertases in rat ovaries during pregnancy. J Ovarian Res 2013 Dec 11;6(1):91.
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