The affinity of human RANK binding to its ligand RANKL.
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 x 10(-10) M.
The Center for Molecular Immunology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
Shiqian
S
Zhang
Changzhen
C
Liu
Peng
P
Huang
Shu
S
Zhou
Jingshan
J
Ren
Yoshihiro
Y
Kitamura
Peifu
P
Tang
Zhenggang
Z
Bi
Bin
B
Gao
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D053245:RANK Ligand / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053246:Receptor Activator of Nuclear Factor-kappa B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance
D017931:DNA Primers; D008024:Ligands; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D011994:Recombinant Proteins; C505686:TNFRSF11A protein, human; C505688:TNFSF11 protein, human