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References

  1. Anderson DM, Maraskovsky E, Billingsley WL, Dougall WC, Tometsko ME, Roux ER, et al. 1997. A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 390: 175-179.
    Pubmed CrossRef
  2. Boyce BF, Xing L. 2008. Functions of RANKL/RANK/OPG in bone modeling and remodeling. Arch. Biochem. Biophys. 473: 139-146.
    Pubmed PMC CrossRef
  3. Fata JE, Kong YY, Li J, Sasaki T, Irie-Sasaki J, Moorehead RA, et al. 2000. The osteoclast differentiation factor osteoprotegerin-ligand is essential for mammary gland development. Cell 103: 41-50.
    CrossRef
  4. Gallagher JC, Riggs BL, Eisman J. 1994. Diagnosis, prophylaxis, and treatment of osteoporosis. Am. J. Med. 90:646-650.
  5. Grigoriades AE, Wang ZQ, Cecchini MG, Hofstetter W, Felix R, Fleisch HA, Wagner EF. 1994. c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling. Science 266: 443-448.
    CrossRef
  6. Ilich JZ, Kerstetter JE. 2000. Nutrition in bone health revisited: a story beyond calcium. J. Am. Coll. Nutr. 19: 715-737.
    Pubmed CrossRef
  7. Jeong TH, Son YJ, Ryu HB, Koo BK, Jeong SM, Hoang P, et al. 2014. Soluble expression and partial purification of recombinant human erythropoietin from E. coli. Protein Expr. Purif. 95: 211-218.
    Pubmed CrossRef
  8. Kanamaru F, Iwai H, Ikeda T, Nakajima A, Ishikawa I, Azuma M. 2004. Expression of membrane-bound and soluble receptor activator of NF-kappa B ligand (RANKL) in human T cells. Immun. Lett. 94: 239-246.
    Pubmed CrossRef
  9. Kim NS, Kim HJ, Koo BK, Kwon MC, Kim YW, Cho Y, et al. 2006. Receptor activator of NF-kappaB ligand regulates the proliferation of mammary epithelial cells. Mol. Cell. Biol. 26:1002-1013.
    Pubmed PMC CrossRef
  10. Kong YY, Yoshida H, Sarosi I, Tan HL, Timms E, Capparelli C, et al. 1999. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 397: 315-323.
    Pubmed CrossRef
  11. O’Brien CA, Nakashima T, Takayanagi H. 2012. Osteocyte control of osteoclastogenesis. Bone 54: 258-263.
    Pubmed PMC CrossRef
  12. Smith DM, Nance WE, Kang KW. 1973. Genetic factors in determining bone mass. J. Clin. Invest. 52: 2800-2808.
    Pubmed PMC CrossRef
  13. Wardlaw G. 1988. The effects of diet and life-style on bone mass in women. J. Am. Diet. Assoc. 88: 17-25.
    Pubmed
  14. Willard D, Chen WJ, Barrett G, Blackburn K, Bynum J, Consler T, et al. 2000. Expression, purification, and characterization of the human receptor activator of NF-KB ligand (RANKL) extracellular domain. Protein Expr. Purif. 20: 48-57.
    Pubmed CrossRef
  15. Wong BR, Rho J, Arron J, Robinson E, Orlinick J, Chao M, et al. 1997. TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells. J. Biol. Chem. 272: 25190-25194.
    Pubmed CrossRef
  16. Xiong J, Charles AO. 2012. Osteocyte RANKL: new insights into t he c ontrol of bone r emodel ing. J. Bone Miner. Res. 27:499-505.
    Pubmed PMC CrossRef

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Article

Research article

J. Microbiol. Biotechnol. 2015; 25(2): 274-279

Published online February 28, 2015 https://doi.org/10.4014/jmb.1407.07002

Copyright © The Korean Society for Microbiology and Biotechnology.

Soluble Expression and Purification of Receptor Activator of Nuclear Factor-Kappa B Ligand Using Escherichia coli

Sol-Ji Park 1, Se-Hoon Lee 2, Kwang-Jin Kim 2, Sung-Gun Kim 3, Hangun Kim 2, Han Choe 4, Sang Yeol Lee 5, Jung-Mi Yun 6, Jae Youl Cho 7, Jiyeon Chun 1, Kap Seong Choi 1 and Young-Jin Son 2*

1Department of Food Science and Technology, Sunchon National University, Suncheon 540-742, Republic of Korea, 2Department of Pharmacy, Sunchon National University, Suncheon 540-742, Republic of Korea, 3Department of Biomedical Science, Youngdong University, Yeongdong-gun, Chungbuk 370-701, Republic of Korea, 4Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul 138-736, Republic of Korea, 5Department of Life Science, Gachon University, Seongnam 461-701, Republic of Korea, 6Department of Food and Nutrition, Kwangju Women’s University, Gwangju 506-713, Republic of Korea, 7Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea

Received: July 2, 2014; Accepted: September 23, 2014

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.

Keywords: RANKL, Osteoclast, Purification, Chromatography, E.coli

References

  1. Anderson DM, Maraskovsky E, Billingsley WL, Dougall WC, Tometsko ME, Roux ER, et al. 1997. A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 390: 175-179.
    Pubmed CrossRef
  2. Boyce BF, Xing L. 2008. Functions of RANKL/RANK/OPG in bone modeling and remodeling. Arch. Biochem. Biophys. 473: 139-146.
    Pubmed KoreaMed CrossRef
  3. Fata JE, Kong YY, Li J, Sasaki T, Irie-Sasaki J, Moorehead RA, et al. 2000. The osteoclast differentiation factor osteoprotegerin-ligand is essential for mammary gland development. Cell 103: 41-50.
    CrossRef
  4. Gallagher JC, Riggs BL, Eisman J. 1994. Diagnosis, prophylaxis, and treatment of osteoporosis. Am. J. Med. 90:646-650.
  5. Grigoriades AE, Wang ZQ, Cecchini MG, Hofstetter W, Felix R, Fleisch HA, Wagner EF. 1994. c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling. Science 266: 443-448.
    CrossRef
  6. Ilich JZ, Kerstetter JE. 2000. Nutrition in bone health revisited: a story beyond calcium. J. Am. Coll. Nutr. 19: 715-737.
    Pubmed CrossRef
  7. Jeong TH, Son YJ, Ryu HB, Koo BK, Jeong SM, Hoang P, et al. 2014. Soluble expression and partial purification of recombinant human erythropoietin from E. coli. Protein Expr. Purif. 95: 211-218.
    Pubmed CrossRef
  8. Kanamaru F, Iwai H, Ikeda T, Nakajima A, Ishikawa I, Azuma M. 2004. Expression of membrane-bound and soluble receptor activator of NF-kappa B ligand (RANKL) in human T cells. Immun. Lett. 94: 239-246.
    Pubmed CrossRef
  9. Kim NS, Kim HJ, Koo BK, Kwon MC, Kim YW, Cho Y, et al. 2006. Receptor activator of NF-kappaB ligand regulates the proliferation of mammary epithelial cells. Mol. Cell. Biol. 26:1002-1013.
    Pubmed KoreaMed CrossRef
  10. Kong YY, Yoshida H, Sarosi I, Tan HL, Timms E, Capparelli C, et al. 1999. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 397: 315-323.
    Pubmed CrossRef
  11. O’Brien CA, Nakashima T, Takayanagi H. 2012. Osteocyte control of osteoclastogenesis. Bone 54: 258-263.
    Pubmed KoreaMed CrossRef
  12. Smith DM, Nance WE, Kang KW. 1973. Genetic factors in determining bone mass. J. Clin. Invest. 52: 2800-2808.
    Pubmed KoreaMed CrossRef
  13. Wardlaw G. 1988. The effects of diet and life-style on bone mass in women. J. Am. Diet. Assoc. 88: 17-25.
    Pubmed
  14. Willard D, Chen WJ, Barrett G, Blackburn K, Bynum J, Consler T, et al. 2000. Expression, purification, and characterization of the human receptor activator of NF-KB ligand (RANKL) extracellular domain. Protein Expr. Purif. 20: 48-57.
    Pubmed CrossRef
  15. Wong BR, Rho J, Arron J, Robinson E, Orlinick J, Chao M, et al. 1997. TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells. J. Biol. Chem. 272: 25190-25194.
    Pubmed CrossRef
  16. Xiong J, Charles AO. 2012. Osteocyte RANKL: new insights into t he c ontrol of bone r emodel ing. J. Bone Miner. Res. 27:499-505.
    Pubmed KoreaMed CrossRef