| Autor: |
Willard, Derril, Chen, Wen-Ji, Barrett, George, Blackburn, Kevin, Bynum, Jane, Consler, Thomas, Hoffman, Christine, Horne, Earnest, Iannone, Marie A., Kadwell, Sue, Parham, Janet, Ellis, Byron |
| Zdroj: |
Protein Expression and Purification; October 2000, Vol. 20 Issue: 1 p48-57, 10p |
| Abstrakt: |
Receptor activator of NF-κB 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 nicell 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-α. 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. |
| Databáze: |
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