PURIFICATION OF MILLIGRAM QUANTITIES OF HUMAN LEPTIN FROM RECOMBINANTESCHERICHIA-COLI

Leptin, the product of the obese (ob) gene, is a 16 kilodalton protein secreted from adipose tissue. Restoration of leptin to obese ob/ob mi ce leads to normalization of body weight. The effect of leptin in larg er animals has not been explored, in part because of limited supplies of leptin. To date, the potency and yield of recombinant leptin purifi ed from a variety of eukaryotic sources or from E. coli has been highl y variable. While purification of leptin from E. coli inclusion bodies has afforded the greatest yield of protein, its potency is at least a n order of magnitude lower than that of leptin secreted from E. coli o r eukaryotic cells. The mechanistic basis of this difference in potenc y is not clear at present. The ability to purify significant quantitie s of highly active leptin will be crucial for the evaluation of leptin structure, as well as its function in additional animal models of obe sity. We now report a facile protocol for the preparation of recombina nt leptin using an E. coli expression system. 75-85 milligrams of lept in with a purity of greater than 97 % was prepared from a liter of rec ombinant E. coli. The procedure can be performed in less than 48 h and requires no chromatography. Intraperitoneal injection of 0.1 mg/kg re natured leptin into ob/ob mice results in a significant reduction in f ood consumption. The potency of this material is similar to the most p otent recombinant leptin described to date. The ability to rapidly pre pare large quantities of high specific activity material will hasten t he definition of leptin's role in non-rodent models of obesity.