Premium
Preparation of Leptin Antagonists by Site‐Directed Mutagenesis of Human, Ovine, Rat, and Mouse Leptin's Site III
Author(s) -
SOLOMON GILI,
NIVSPECTOR LEONORA,
GONENBERGER DANA,
CALLEBAUT ISABELLE,
DJIANE JEAN,
GERTLER ARIEH
Publication year - 2006
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1196/annals.1378.094
Subject(s) - chemistry , size exclusion chromatography , affinity chromatography , biochemistry , site directed mutagenesis , pegylation , ion chromatography , biological activity , receptor , cysteine , mutagenesis , microbiology and biotechnology , in vitro , enzyme , biology , mutant , polyethylene glycol , gene
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 .