Endocrine Abstracts

Background: Leptin is an important regulator of the immune response. Therapeutics that modulate leptin signalling are considered to be a potential targets in a number of immune related conditions. The role of leptin antagonists to suppress the immune system, slowing the progression of autoimmune diseases is a widely discussed idea. However, leptin agonists may also be useful to up-regulate the immune response during malnutrition. The aim of this project was to develop long-lasting leptin agonists, using ligand–receptor fusion technology (LR).

Method: The LR fusion was generated by linking leptin to the leptin binding domain (LBD) of the human leptin receptor. The molecules were joined by a flexible glycine–serine linker. The chimeric protein was expressed as insoluble inclusion bodies in E. coli. The protein was refolded and purified by ion exchange and gel filtration chromatography. The bioactivity was assessed by an in-house duel luciferase reporter assay for STAT-3 activity, in cells expressing the full length human leptin receptor.

Results: The LR agonist expressed well in E. coli, but formed insoluble inclusion bodies. Refolding and purification of the chimera resulted in a 42KDa protein that was >90% pure by SDS-PAGE analysis. Initial testing of bioactivity showed activation of leptin signalling pathway at approximately half the level of standard leptin.

Conclusions: We have expressed and purified a recombinant leptin agonist, based on a ligand–receptor fusion. Initial characterisation of the chimera revealed good bioactivity. Further in vitro mouse studies are planned to determine if the chimera has a longer serum half-life than leptin.