title: Preparation of leptin antagonists by site-directed mutagenesis of human, ovine, rat, and mouse leptin's site III: implications on blocking undesired leptin action in vivo.
abstract: Six muteins of human, ovine, rat, and mouse leptins mutated to Ala in amino acids 39-41 or 39-42 were prepared by site-directed mutagenesis of the putative site III, which does not affect binding but is necessary for receptor activation, then expressed, solubilized in 4.5 M urea, at pH 11.3 in presence of cysteine, refolded and purified to homogeneity by anion-exchange chromatography on Q-Sepharose or combination of anion-exchange chromatography followed by gel filtration. The overall yields were 400-800 mg from 5 L of fermentation. All proteins were >98% pure as evidenced by SDS-PAGE and contained at least 95% monomers as documented by gel-filtration chromatography under nondenaturing conditions. Circular dichroism analysis revealed that all six muteins have identical secondary structure characteristic of nonmutated leptins, namely 52-63% of alpha helix content. All muteins formed a 1:1 complex with chicken leptin binding domain, (chLBD) and bound chLBD or membrane-embedded leptin receptor with affinity identical to WT leptins. Muteins were devoid of any biological activity in several bioassays but were potent competitive antagonists. Some muteins were pegylated using 40 kDa PEG. Although pegylation decreased the in vitro activity, increasing circulation half-life can recompensate this deficit, so pegylated antagonists are expected to be more potent in vivo.
authors.affiliation: Faculty of Agricultural, Food, and Environmental Quality Sciences, The Hebrew University of Jerusalem, POB 12, Rehovot 76100, Israel.
authors.forename: Gili
authors.initials: G
authors.lastname: Solomon
authors.forename: Leonora
authors.initials: L
authors.lastname: Niv-Spector
authors.forename: Dana
authors.initials: D
authors.lastname: Gonen-Berger
authors.forename: Isabelle
authors.initials: I
authors.lastname: Callebaut
authors.forename: Jean
authors.initials: J
authors.lastname: Djiane
authors.forename: Arieh
authors.initials: A
authors.lastname: Gertler
mesh_terms: D000818:Animals; D002460:Cell Line; D002645:Chickens; D006801:Humans; D020738:Leptin / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D012756:Sheep
chemical_list: D020738:Leptin