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Expression and bioactivity of a single chain recombinant equine luteinizing hormone (reLH).
- Source :
-
Theriogenology [Theriogenology] 2007 Jan 15; Vol. 67 (2), pp. 311-20. Date of Electronic Publication: 2006 Oct 17. - Publication Year :
- 2007
-
Abstract
- To study structure-activity relationships and the role of equine gonadotropins in the normal and pathophysiology of equine reproduction, the availability of purified hormones is essential. Previous expression studies in transfected CHO cells showed inefficient assembly of the human and bovine alpha and beta subunits, resulting in low levels of recombinant LH. The ability to express a single chain bearing genetically linked alpha and beta subunits bypasses this rate-limiting assembly step. A chimera was constructed by overlap PCR in which the carboxy terminal end of the eLHbeta subunit was genetically fused to the amino end of the alpha subunit. This gene was transfected into CHO cells and the recombinant product was purified through multiple steps, including a Fractogel resin separation. Serial dilutions of pituitary derived native eLH and the single chain reLH were compared in an eLH radioimmunoassay (RIA); the concentration curves between the single chain recombinant eLH and the native eLH standard were parallel. The biological activity of the analog was determined in vitro and in vivo using homologous equine models. Testicular tissue from five colts was processed for Leydig cell cultures. Increasing doses of reLH were incubated with equine Leydig cells for 24h in vitro and testosterone production was determined by RIA. Recombinant eLH stimulated a greater than 15-fold increase in testosterone production in a dose-dependent manner. Quarter Horse breeding stallions were treated with either reLH (n=5) or saline (n=3) and plasma testosterone concentrations were measured by RIA. Recombinant eLH stimulated a four-fold increase in circulating testosterone concentrations compared to the saline control. Therefore, the single chain recombinant will be effective for a variety of structure-function analyses and for breeding management in the horse.
- Subjects :
- Animals
CHO Cells
Chimera
Cricetinae
Cricetulus
Dose-Response Relationship, Drug
Horses genetics
Leydig Cells drug effects
Luteinizing Hormone chemistry
Luteinizing Hormone genetics
Luteinizing Hormone pharmacology
Male
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins metabolism
Recombinant Proteins pharmacology
Structure-Activity Relationship
Transfection veterinary
Horses physiology
Leydig Cells metabolism
Luteinizing Hormone metabolism
Testosterone biosynthesis
Subjects
Details
- Language :
- English
- ISSN :
- 0093-691X
- Volume :
- 67
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Theriogenology
- Publication Type :
- Academic Journal
- Accession number :
- 17049590
- Full Text :
- https://doi.org/10.1016/j.theriogenology.2006.06.013