The affinity of human RANK binding to its ligand RANKL

• Published in: Archives of biochemistry and biophysics Vol. 487; no. 1; pp. 49 - 53
• Main Authors: Zhang, Shiqian, Liu, Changzhen, Huang, Peng, Zhou, Shu, Ren, Jingshan, Kitamura, Yoshihiro, Tang, Peifu, Bi, Zhenggang, Gao, Bin
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.07.2009
• Subjects: Affinity of RANK–RANKL; Base Sequence; Bone; Chromatography; Digestion; DNA Primers - genetics; Enzymes; Escherichia coli; Escherichia coli - genetics; Filtration; Fusion protein; Humans; Immune response; In Vitro Techniques; Inclusion bodies; Kinetics; Ligands; Mammary gland; Monomers; NF- Kappa B protein; Protein Interaction Domains and Motifs; Protein Structure, Tertiary; RANK ligand; RANK Ligand - chemistry; RANK Ligand - genetics; RANK Ligand - metabolism; RANK–RANKL interaction; Receptor activator of nuclear factor-kappa B; Receptor Activator of Nuclear Factor-kappa B - chemistry; Receptor Activator of Nuclear Factor-kappa B - genetics; Receptor Activator of Nuclear Factor-kappa B - metabolism; Receptor mechanisms; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Surface Plasmon Resonance; TRANCE protein; Vascular diseases; RANK–RANKL interaction; Affinity of RANK–RANKL; RANK ligand; Receptor activator of nuclear factor-kappa B; Surface plasmon resonance
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2009.04.008

Receptor activator of nuclear factor-kappa B (RANK) and its ligand, RANKL play critical roles in bone re-modeling, immune function, vascular disease and mammary gland development. To study the interaction of RANK and RANKL, we have expressed both extracellular domain of RANK and ectodomain of RANKL using Escherichia coli expression system. RANK was expressed as an inclusion body first which properly refolded later, while RANKL was initially produced as a GST fusion protein, after which the GST was removed by enzyme digestion. Soluble RANK existed as a monomer while RANKL was seen as a trimer in solution, demonstrated by gel filtration chromatography and cross-linking experiment. The recombinant RANK and RANKL could bind to each other and the binding affinity of RANKL for RANK was measured with surface plasmon resonance technology and K D value is about 1.09 × 10 −10 M.