Large-scale preparation of biologically active recombinant ovine obese protein (leptin).
Prokaryotic expression vector pMON3401 encoding full size A(-1) ovine leptin was prepared by polymerase chain reaction (PCR) of previously described cDNA. E. coli cells transformed with this vector overexpressed large amounts of ovine leptin upon induction with nalidixic acid. The expressed protein found in the inclusion bodies was refolded and purified to homogeneity on Q-Sepharose and SP-Sepharose columns, yielding two electrophoretically pure fractions (leptin-Q and leptin-SP), composed respectively of 90 and 95% of monomeric protein of the expected molecular mass of 16 kDa. The purified protein was capable of interacting with antibodies raised against (GST-ovine leptin and to bind specifically to ventromedial hypothalamus of ewes. The biological activity of both fractions resulting from proper renaturation was further evidenced by their ability to stimulate DNA synthesis in leptin-sensitive BAF/3 cells transfected with a long form of human leptin receptor construct.
Gertler
A
A
Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, Israel. gertler@agri.huji.ac.il
Simmons
J
J
Keisler
D H
DH
D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli; D006801:Humans; D007031:Hypothalamus / Q000378:metabolism; D020738:Leptin; D051379:Mice; D009765:Obesity; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry / Q000494:pharmacology*; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology; D012756:Sheep; D014162:Transfection
D002352:Carrier Proteins; C515448:LEPR protein, human; D020738:Leptin; D011506:Proteins; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D011994:Recombinant Proteins; C515447:leptin receptor, mouse