Endocrine Abstracts

Introduction: Leptin is a 16-kDa peptide hormone secreted by adipose tissue and acts as a sensor for energy stores. It feedsback at the hypothalamic arcuate nucleus to suppress appetite. Leptin treatment has been highly effective in suppressing appetite in the rare cases of leptin-deficient obesity and improving the metabolic profile in congenital generalised lipodystrophy. These patients require 2.5–10 mg once daily recombinant leptin treatment. We hypothesised that prolonged constant exposure to leptin with a long acting leptin may have a more potent metabolic action and appetite supression and therefore we designed a long acting leptin molecule.

Aim: To construct, express, purify and test for bioactivity a long acting leptin.

Methods: We have previously demonstrated that a fusion of growth hormone to growth hormone binding protein (GHBP) generated a long acting GH. In this project we utilise GHBP as a fusion partner with leptin the concept being that linking a fusion protein to the C-terminus will decrease clearance through reduced proteolysis and renal clearance. We further modified this molecule by introducing a W104A (Tryptophan-Alanine amino acid substitution) in the GHBP to prevent GHBP binding to GH in the circulation.

Results: GHBP(W104A)-leptin fusion was cloned in to a modified invitrogen pSecTag plasmid with the secretion sequence for leptin. This plasmid was transfected into CHO Flp-In cells by reagent-mediated transfection. Protein was expressed in CHO cells grown in roller bottles in the presence of valproic acid at a set temperature of 31 °C and purified by antibody affinity chromatography. In vitro bioactivity was assessed by luciferase expression induced by leptin. Approximately two times fold induction was achieved.

Conclusion: We have demonstrated it is possible to generate a fusion of leptin to GHBP and that this fusion retains bioactivity. Future studies will assess pharmacodynamic and pharmacokinetic properties of this molecule.