Recombinant expression of biologically active rat leptin in Escherichia coli

Leptin is a 16-kDa nonglycosylated hormone that is produced in mature adipo cytes and which acts primarily in the hypothalamus to reduce food intake an d body weight. While the rat is a representative laboratory animal model in obesity research, so far recombinant rat leptin was not available. In the present study, rat leptin was recombinantly expressed in Escherichia coli a nd purified in a bioactive form to provide a further tool for the analysis of leptin functions in rats. Leptin cDNA was cloned by RT-PCR from total RN A of SD rat adipocytes, and overexpression was achieved by subcloning the l eptin cDNA into the pET-29a vector, which enabled the recombinant expressio n of rat leptin as an S-peptide-tagged fusion protein. Since the fusion pro teins were expressed in inclusion bodies, after purification of the insolub le fraction, leptin proteins were refolded by sequential dialysis into phys iological buffers. The biological activity of this recombinant protein was confirmed in proliferation assays using leptin-sensitive rat insulinoma cel ls as well as a newly developed leptin-sensitive luciferase assay system. T he specific binding of the S-tagged leptin to leptin-receptor-expressing ce lls was further shown by flow cytometry using fluorescence-conjugated S-pro teins. (C) 2001 Academic Press.