Production of Leptin inEscherichia coli:A Comparison of Methods |
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| Authors: | Jeffrey P. Varnerin Timothy Smith Charles I. Rosenblum Aurawan Vongs Beth A. Murphy Chris Nunes Theodore N. Mellin Joseph J. King Bruce W. Burgess Beth Junker Michael Chou Patricia Hey Easter Frazier D.Euan MacIntyre Lex H.T. Van der Ploeg Michael R. Tota |
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| Affiliation: | aDepartment of Obesity Research, Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey, 07065;bDepartment of Pharmacology, Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey, 07065;cDepartment of Bioprocess Research and Development, Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey, 07065 |
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| Abstract: | A procedure is described for gram-scale refolding ofEscherichia coli-derived human leptin inclusion bodies. Refolding was achieved by gradually reducing denaturant using a diafiltration method. Refolded leptin is characterized byin vivomodulation of food intake, reduction in body weight, and lowering of insulin and glucose levels inob/obmice. In addition, refolded leptin is characterized by radioimmunoassay (RIA) and activation of the leptin receptor in a cell-based assay. For comparison we also refolded leptin by a simple dilution method and produced periplasmic derived leptin, which did not requireex vivofolding. Leptin produced by these three methods and leptin obtained from commercial sources were compared using the RIA and the cell-based assay and appeared to be of comparable quality and potency. |
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