Bacterial production and functional characterization of the satiety hormone leptin as a potential therapeutic agent with prolonged plasma half-life

Leptin, a 16 kDa (146 amino acids) peptide hormone, plays a central role in the control of metabolism and appetite. Its clinical application in diabetes type II has been proposed. However, due to its small size leptin is rapidly eliminated by renal clearance. With a very short plasma half-life in humans, at least daily injections would be necessary, which is not acceptable as a standard therapy.

In this work we have enlarged the hydrodynamic volume of mouse leptin by PASylation: fusion with a poly amino acid sequence comprising proline, alanine and serine (PAS) residues at either the N- or the C-terminus. Six fusion proteins with 200 (PAS200), 400 (PAS400) or 600 (PAS600) PAS residues were constructed and produced. With their PEG-like behavior the PAS tags are expected to increase the biomolecular size of the protein beyond the pore size of the kidney, thus prolonging blood circulation. Indeed, analytical size exclusion chromatography showed a dramatic increase of the hydrodynamic volume: 5x for the PAS200, 10x for the PAS400 and 13x for the PAS600 fusion proteins. Circular dichroism spectroscopy proved that the α-helical structure of native leptin is accompanied by a random coil structure of the PAS moiety, getting more prominent with growing chain length. As demonstrated in SPR measurements, the KD of the fusion proteins to the leptin receptor is almost fully retained.

Some of the engineered variants have been tested in a cell culture assay using mObR-transfected HEK293 cells harbouring a STAT3-responsive luciferase reporter gene and shown to be almost as effective as the unfused recombinant leptin. In vivo investigation of their pharmaceutical properties in mice is underway.