Expression and bioactivity of a single chain recombinant equine luteinizing hormone (reLH).
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.
Publication Date: 2006-10-17 PubMed ID: 17049590DOI: 10.1016/j.theriogenology.2006.06.013Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research article revolves around the creation and testing of a single chain recombinant equine luteinizing hormone (reLH). It focuses on determining the impact of equine gonadotropins in normal and abnormal conditions of equine reproduction with the help of purified hormones. The researchers successfully created a chimera that can potentially be beneficial for analysis of structure-function and breeding management in horses.
Creation of the Recombinant Equine Luteinizing Hormone
- The researchers used a technique, called overlap PCR, to develop a chimera: they fused the carboxy terminal end of the eLHbeta subunit to the amino end of the alpha subunit. It resulted in the formation of a single chain that bears both alpha and beta subunits of the LH.
- This new gene was then transfected into CHO cells to express the linked alpha and beta subunits. This process bypasses the rate-limiting assembly step that often leads to low levels of recombinant LH.
- The recombinant product was purified through several steps, culminating in a Fractogel resin separation.
Comparison with Native eLH
- Serial dilutions were prepared of the native eLH (extracted from the pituitary gland) and the recombinant eLH for comparison.
- An eLH radioimmunoassay was used to analyze and identify the similarities between the single chain recombinant eLH and native eLH. The resulting concentration curves were found to be parallel.
The Biological Activity of the Recombinant eLH
- In vitro tests: Leydig cells derived from testicular tissue of colts were treated with increasing doses of reLH for 24 hours. This was followed by determining the testosterone production. The results showed a more than 15-fold increase in testosterone production, dependent on the reLH dosage.
- In vivo tests: Quarter Horse breeding stallions were treated with either reLH or saline. Plasma testosterone concentrations were measured, and the results showed a four-fold increase in circulating testosterone concentrations when treated with recombinant eLH compared to the saline control.
Conclusion
- The single-chain recombinant equine luteinizing hormone promises to be effective for various structure-function analyses. Additionally, it seems to be beneficial for managing breeding in horses.
Cite This Article
APA
Jablonka-Shariff A, Roser JF, Bousfield GR, Wolfe MW, Sibley LE, Colgin M, Boime I.
(2006).
Expression and bioactivity of a single chain recombinant equine luteinizing hormone (reLH).
Theriogenology, 67(2), 311-320.
https://doi.org/10.1016/j.theriogenology.2006.06.013 Publication
Researcher Affiliations
- Department of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 South Euclid Avenue Box 8103, St. Louis, MO 63110, USA.
MeSH Terms
- Animals
- CHO Cells
- Chimera
- Cricetinae
- Cricetulus
- Dose-Response Relationship, Drug
- Horses / genetics
- Horses / physiology
- Leydig Cells / drug effects
- Leydig Cells / metabolism
- Luteinizing Hormone / chemistry
- Luteinizing Hormone / genetics
- Luteinizing Hormone / metabolism
- Luteinizing Hormone / pharmacology
- Male
- Recombinant Proteins / chemistry
- Recombinant Proteins / genetics
- Recombinant Proteins / metabolism
- Recombinant Proteins / pharmacology
- Structure-Activity Relationship
- Testosterone / biosynthesis
- Transfection / veterinary
Citations
This article has been cited 3 times.- Gifre L, Arís A, Bach À, Garcia-Fruitós E. Trends in recombinant protein use in animal production.. Microb Cell Fact 2017 Mar 4;16(1):40.
- Cohen L, Bousfield GR, Ben-Menahem D. The recombinant equine LHβ subunit combines divergent intracellular traits of human LHβ and CGβ subunits.. Theriogenology 2015 Jun;83(9):1469-76.
- Cahoreau C, Klett D, Combarnous Y. Structure-function relationships of glycoprotein hormones and their subunits' ancestors.. Front Endocrinol (Lausanne) 2015;6:26.
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