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J. Biol. Chem., Vol. 277, Issue 48, 46304-46309, November 29, 2002

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Subcloning, Expression, Purification, and Characterization of Recombinant Human Leptin-binding Domain^*

Yael Sandowski^§, Nina Raver^§¶, Eugene E. Gussakovsky, Suzan Shochat^**, Orly Dym^**, Oded Livnah^**, Menachem Rubinstein, Radha Krishna, and Arieh Gertler^¶§§

From the ^¶ Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University, Rehovot 76100, Israel,  Department of Life Sciences, Bar Ilan University, Ramat Gan 52900, Israel and Institute of Horticulture, The Volcani Center, ARO, Bet Dagan 50250, Israel, ^** The Institute of Life Sciences, Faculty of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel,  Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel and  Diagnostic System Laboratories, Webster, Texas 77598

A subdomain of the human leptin receptor encoding part of the extracellular domain (amino acids 428 to 635) was subcloned, expressed in a prokaryotic host, and purified to homogeneity, as evidenced by SDS-PAGE, with over 95% monomeric protein. The purified leptin-binding domain (LBD) exhibited the predicted  structure, was capable of binding human, ovine, and chicken leptins, and formed a stable 1:1 complex with all mammalian leptins. The binding kinetics, assayed by surface plasmon resonance methodology, showed respective k_on and k_off values (mean ± S.E.) of 1.20 ± 0.23 × 10⁵ mol¹ s¹ and 1.85 ± 0.30 × 10³ s¹ and a K_d value of 1.54 × 10⁸ M. Similar results were achieved with conventional binding experiments. LBD blocked leptin-induced, but not interleukin-3-induced, proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. The modeled LBD structure and the known three-dimensional structure of human leptin were used to construct a model of 1:1 LBD·human leptin complex. Two main residues, Phe-500, located in loop L3, and Tyr-441, located in L1, are suggested to contribute to leptin binding.

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