SFEBES2008 Poster Presentations Cytokines and growth factors (7 abstracts)
Background: Leptin has multiple functions including modulating energy homeostasis and the immune response. Leptin is an important target for therapy to modulate the immune response in several autoimmune diseases. Although the structure of leptin is known, that of its receptor remains unsolved. The aim of this project was to express and purify the extracellular domain the leptin receptor (ObR) in order to carry out functional and crystallisation studies to elucidate the mechanisms of leptin binding.
Methods: We expressed and characterised the 25 kDa leptin-binding domain (LBD) of ObR in E. coli. We used this purified LBD to screen a panel of anti-ObR mouse monoclonal antibodies (MAbs). A mammalian expression system was used to express the full extracellular domain of ObR; expression levels were determined by an in-house ELISA.
Results: The LBD was expressed in E. coli as insoluble inclusion bodies. These were solubilised in urea, refolded by dialysis and purified by one-step anion exchange chromatography. However concentrating LBD above 200 μg/ml was hindered by protein aggregation. Purified LBD was shown to bind leptin at high affinity (128 nM) forming a stable 1:1 complex that could be further purified by gel filtration chromatography. The MAb screen identified several antibodies that bound LBD with high affinity. The full extracellular domain of ObR was expressed in HEK 293 cells at a level of 100 ng/ml and a small quantity was purified by antibody affinity chromatography.
Conclusions: We have expressed and purified the full extracellular domain of ObR in mammalian cells. Expression levels were very low thus further work is required to optimise expression. The LBD was expressed in E. coli and shown to bind leptin and several MAbs with high affinity. Characterising the crystal structure of ObR may allow rational design of antagonists for the treatment of several autoimmune diseases.