title: Soluble expression and purification of receptor activator of nuclear factor-kappa B ligand using Escherichia coli.
abstract: Receptor activator of nuclear factor-kappa B ligand (RANKL) is a critical factor in osteoclastogenesis. It makes osteoclasts differentiate and multinucleate in bone remodeling. In the present study, RANKL was expressed as a soluble maltose binding protein (MBP)-fusion protein using the Escherichia coli maltose binding domain tag system (pMAL) expression vector system. The host cell E. coli DH5α was cultured and induced by isopropyl β-D-1- thiogalactopyranoside for rRANKL expression. Cells were disrupted by sonication to collect soluble MBP-fused rRANKL. The MBP-fusion rRANKL was purified with MBP Trap affinity chromatography and treated with Tobacco Etch Virus nuclear inclusion endopeptidase (TEV protease) to remove the MBP fusion protein. Dialysis was then carried out to remove binding maltose from the cleaved rRANKL solution. The cleaved rRANKL was purified with a second MBP Trap affinity chromatography to separate unsevered MBP-fusion rRANKL and cleaved MBP fusion protein. The purified rRANKL was shown to have biological activity by performing in vitro cell tests. In conclusion, biologically active rRANKL was successfully purified by a simple two-step chromatography purification process with one column.
authors.affiliation: Department of Food Science and Technology, Sunchon National University, Suncheon 540-742, Republic of Korea.
authors.forename: Sol-Ji
authors.initials: SJ
authors.lastname: Park
authors.forename: Se-Hoon
authors.initials: SH
authors.lastname: Lee
authors.forename: Kwang-Jin
authors.initials: KJ
authors.lastname: Kim
authors.forename: Sung-Gun
authors.initials: SG
authors.lastname: Kim
authors.forename: Hangun
authors.initials: H
authors.lastname: Kim
authors.forename: Han
authors.initials: H
authors.lastname: Choe
authors.forename: Sang Yeol
authors.initials: SY
authors.lastname: Lee
authors.forename: Jung-Mi
authors.initials: JM
authors.lastname: Yun
authors.forename: Jae Youl
authors.initials: JY
authors.lastname: Cho
authors.forename: Jiyeon
authors.initials: J
authors.lastname: Chun
authors.forename: Kap Seong
authors.initials: KS
authors.lastname: Choi
authors.forename: Young-Jin
authors.initials: YJ
authors.lastname: Son
mesh_terms: D002846:Chromatography, Affinity; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D008264:Macrophages / Q000502:physiology; D058619:Maltose-Binding Proteins / Q000235:genetics; D053245:RANK Ligand / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification
chemical_list: D058619:Maltose-Binding Proteins; D053245:RANK Ligand; D011993:Recombinant Fusion Proteins; D010450:Endopeptidases; C099488:TEV protease