title: Improved efficacy of soluble human receptor activator of nuclear factor kappa B (RANK) fusion protein by site-directed mutagenesis.
abstract: Soluble human receptor activator of nuclear factor kappa B fusion immunoglobulin (hRANK-Ig) has been considered as one of the therapeutic agents to treat osteoporosis or diseases associated with bone destruction by blocking the interaction between RANK and the receptor activator of nuclear factor kappa B ligand (RANKL). However, no scientific record showing critical amino acid residues within the structural interface between the human RANKL and RANK complex is yet available. In this study, we produced several mutants of hRANK-Ig by replacement of amino acid residue(s) and tested whether the mutants had increased binding affinity to human RANKL. Based on the results from flow cytometry and surface plasmon resonance analyses, the replacement of E(125) with D(125), or E(125) and C(127) with D(125) and F(127) within loop 3 of cysteine-rich domain 3 of hRANK-Ig increases binding affinity to human RANKL over the wild-type hRANK-Ig. This result may provide the first example of improvement in the efficacy of hRANK-Ig by protein engineering and may give additional information to understand a more defined structural interface between hRANK and RANKL.
authors.affiliation: Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University , Seoul , Republic of Korea and.
authors.forename: Young Jun
authors.initials: YJ
authors.lastname: Son
authors.forename: Jihye
authors.initials: J
authors.lastname: Han
authors.forename: Jae Yeon
authors.initials: JY
authors.lastname: Lee
authors.forename: HaHyung
authors.initials: H
authors.lastname: Kim
authors.forename: Taehoon
authors.initials: T
authors.lastname: Chun
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007136:Immunoglobulins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D017433:Protein Structure, Secondary; D053246:Receptor Activator of Nuclear Factor-kappa B / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D012995:Solubility
chemical_list: D007136:Immunoglobulins; D053246:Receptor Activator of Nuclear Factor-kappa B; D011993:Recombinant Fusion Proteins; C505686:TNFRSF11A protein, human
keywords: Immunoglobulin fusion protein; mutagenesis; osteoclast; osteoporosis; receptor activator of nuclear factor kappa B