Purification of milligram quantities of human leptin from recombinant E. 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 mice leads to normalization of body weight. The effect of leptin in larger animals has not been explored, in part because of limited supplies of leptin. To date, the potency and yield of recombinant leptin purified from a variety of eukaryotic sources or from E. coli has been highly variable. While purification of leptin from E. coli inclusion bodies has afforded the greatest yield of protein, its potency is at least an order of magnitude lower than that of leptin secreted from E. coli or eukaryotic cells. The mechanistic basis of this difference in potency is not clear at present. The ability to purify significant quantities of highly active leptin will be crucial for the evaluation of leptin structure, as well as its function in additional animal models of obesity. We now report a facile protocol for the preparation of recombinant leptin using an E. coli expression system. 75-85 milligrams of leptin with a purity of greater than 97 % was prepared from a liter of recombinant E. coil. The procedure can be performed in less than 48 h and requires no chromatography. Intraperitoneal injection of 0.1 mg/kg renatured leptin into ob/ob mice results in a significant reduction in food consumption. The potency of this material is similar to the most potent recombinant leptin described to date. The ability to rapidly prepare large quantities of high specific activity material will hasten the definition of leptin's role in non-rodent models of obesity.
Fawzi
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AB
Dept. of CNS/Cardiovascular Pharmacology, Schering-Plough Research Institute, Kenilworth, NJ 07033-0539, USA.
Zhang
H
H
van Heek
M
M
Graziano
M P
MP
D000818:Animals; D004435:Eating / Q000187:drug effects; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D015870:Gene Expression; D006801:Humans; D007274:Injections, Intraperitoneal; D020738:Leptin; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D051379:Mice; D008820:Mice, Obese; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology
D020738:Leptin; D046911:Macromolecular Substances; D011506:Proteins; D011994:Recombinant Proteins