KMID : 0545120150250020274
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Journal of Microbiology and Biotechnology 2015 Volume.25 No. 2 p.274 ~ p.279
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Soluble Expression and Purification of Receptor Activator of Nuclear Factor-Kappa B Ligand Using Escherichia coli
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Park Sol-Ji
Lee Se-Hoon Kim Kwang-Jin Kim Sung-Gun Kim Han-Gun Choe Han Lee Sang-Yeol Yun Jung-Mi Cho Jae-Youl Chun Ji-Yeon Choi Kap-Seong Son Young-Jin
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Abstract
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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 DH5¥á was cultured and induced by isopropyl ¥â-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.
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KEYWORD
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RANKL, Osteoclast, Purification, Chromatography, E.coli
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