Expression, purification, and characterization of the human receptor activator of NF-kappaB ligand (RANKL) extracellular domain.
Receptor activator of NF-kappaB ligand (RANKL) is a type II transmembrane protein found on osteoblasts which functions as a major determinant of osteoclast differentiation and activation. RANKL mediates bone homeostasis through binding to the cognate ligand on osteoclasts, RANK, and a soluble decoy receptor, osteoprotegerin (OPG). We designed a construct encoding the extracellular domain of human RANKL that conformed to reports of native processing. To encourage folding and posttranslational modification of a normally membrane-inserted moiety, we expressed the RANKL truncate as a secreted protein using the signal sequence from OPG in a Trichoplusia ni cell line using a baculovirus expression vector. RANKL was purified by a three-step process including an OPG-Fc affinity column. SDS-PAGE and mass spectral analysis indicated that the protein was >99% pure and glycosylated. Circular dichroism spectra revealed that the protein exhibited structural elements similar to tumor necrosis factor-alpha. By BIAcore analysis, RANKL bound to OPG with an affinity of 6.7 nM. Sedimentation equilibrium analytical ultracentrifugation analyses established that our protein existed as a trimer. We conclude that our expressed human RANKL truncate is folded, is functional, and exhibits self-association consistent with other family members.
Willard
D
D
Molecular Sciences Department, Structural Chemistry Department, Glaxo Wellcome, Inc., Five Moore Drive, Research Triangle Park, North Carolina 27709, USA. dhw29438@glaxowellcome.com
Chen
W J
WJ
Barrett
G
G
Blackburn
K
K
Bynum
J
J
Consler
T
T
Hoffman
C
C
Horne
E
E
Iannone
M A
MA
Kadwell
S
S
Parham
J
J
Ellis
B
B
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D002460:Cell Line; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D003001:Cloning, Molecular; D006801:Humans; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D014461:Ultracentrifugation
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; C505686:TNFRSF11A protein, human; C505688:TNFSF11 protein, human