Abstract
NELL1 is a secretory protein that induces osteogenic differentiation and bone formation by osteoblastic cells. Because of its potent osteoinductive activity, NELL1 may be useful for bone regeneration therapy. However, at present, we have little knowledge regarding NELL1 receptors and NELL1-mediated signaling pathways. We have previously produced NELL1 using an insect’s cell expression system; however, the protein was relatively unstable and was degraded by proteases released from dead cells. In the present study, NELL1 protein was expressed in human embryonic kidney 293-F cells. Stable cell lines expressing NELL1 fused to a C-terminal hexahistidine-tag were obtained by G418 selection of transfected cells. Cells grown in serum-free medium showed high levels of NELL1 protein production (approximately 4 mg/l cell culture) for up to 6 months. NELL1 protein was purified from culture medium using a one-step nickel-chelate affinity chromatography protocol. Purified NELL1 protein immobilized onto culture dishes induced the expression of both early and late osteogenic markers on mouse mesenchymal C3H10T1/2 cells. When NELL1-expressing 293-F cells were grown on gelatin-coated glass cover slips, recombinant NELL1 was deposited in the extracellular matrix after detachment of cells. These results suggest that NELL1 acts as an extracellular matrix component. Recombinant NELL1 formed multimers and was glycosylated. An abundant source of functionally active NELL1 protein will be useful for more advanced studies, such as the development of novel techniques for bone regeneration.
Similar content being viewed by others
References
Zhang, X., Zara, J., Siu, R. K., Ting, K., & Soo, C. (2010). The role of NELL-1, a growth factor associated with craniosynostosis, in promoting bone regeneration. Journal of Dental Research, 89, 865–878.
Ting, K., Vastardis, H., Mulliken, J. B., Soo, C., Tieu, A., Do, H., et al. (1999). Human NELL-1 expressed in unilateral coronal synostosis. Journal of Bone and Mineral Research, 14, 80–89.
Zhang, X., Kuroda, S., Carpenter, D., Nishimura, I., Soo, C., Moats, R., et al. (2002). Craniosynostosis in transgenic mice overexpressing Nell-1. Journal of Clinical Investigation, 110, 861–870.
Desai, J., Shannon, M. E., Johnson, M. D., Ruff, D. W., Hughes, L. A., Kerley, M. K., et al. (2006). Nell1-deficient mice have reduced expression of extracellular matrix proteins causing cranial and vertebral defects. Human Molecular Genetics, 15, 1329–1341.
Aghaloo, T., Cowan, C. M., Chou, Y. F., Zhang, X., Lee, H., Miao, S., et al. (2006). Nell-1-induced bone regeneration in calvarial defects. American Journal of Pathology, 169, 903–915.
Bokui, N., Otani, T., Igarashi, K., Kaku, J., Oda, M., Nagaoka, T., et al. (2008). Involvement of MAPK signaling molecules and Runx2 in the NELL1-induced osteoblastic differentiation. FEBS Letters, 582, 365–371.
Kuroda, S., Oyasu, M., Kawakami, M., Kanayama, N., Tanizawa, K., Saito, N., et al. (1999). Biochemical characterization and expression analysis of neural thrombospondin-1-like proteins NELL1 and NELL2. Biochemical and Biophysical Research Communications, 265, 79–86.
Cowan, C. M., Cheng, S., Ting, K., Soo, C., Walder, B., Wu, B., et al. (2006). Nell-1 induced bone formation within the distracted intermaxillary suture. Bone, 38, 48–58.
Zhang, X., Cowan, C. M., Jiang, X., Soo, C., Miao, S., Carpenter, D., et al. (2006). Nell-1 induces acrania-like cranioskeletal deformities during mouse embryonic development. Laboratory Investigation, 86, 633–644.
Aghaloo, T., Jiang, X., Soo, C., Zhang, Z., Zhang, X., Hu, J., et al. (2007). A study of the role of nell-1 gene modified goat bone marrow stromal cells in promoting new bone formation. Molecular Therapy, 15, 1872–1880.
Lu, S. S., Zhang, X., Soo, C., Hsu, T., Napoli, A., Aghaloo, T., et al. (2007). The osteoinductive properties of Nell-1 in a rat spinal fusion model. Spine Journal, 7, 50–60.
Aghaloo, T., Cowan, C. M., Zhang, X., Freymiller, E., Soo, C., Wu, B., et al. (2010). The effect of NELL1 and bone morphogenetic protein-2 on calvarial bone regeneration. Journal of Oral and Maxillofacial Surgery, 68, 300–308.
Li, W., Lee, M., Whang, J., Siu, R. K., Zhang, X., Liu, C., et al. (2010). Delivery of lyophilized Nell-1 in a rat spinal fusion model. Tissue Engineering Part A, 16, 2861–2870.
Siu, R. K., Lu, S. S., Li, W., Whang, J., McNeill, G. R., Zhang, X., et al. (2011). Nell-1 protein promotes bone formation in a sheep spinal fusion model. Tissue Engineering Part A, 17, 1123–1135.
Xue, J., Peng, J., Yuan, M., Wang, A., Zhang, L., Liu, S., et al. (2011). NELL1 promotes high-quality bone regeneration in rat femoral distraction osteogenesis model. Bone, 48, 485–495.
Zhang, X., Ting, K., Bessette, C. M., Culiat, C. T., Sung, S. J., Lee, H., et al. (2011). Nell-1, a key functional mediator of Runx2, partially rescues calvarial defects in Runx2(±) mice. Journal of Bone and Mineral Research, 26, 777–791.
Cowan, C. M., Jiang, X., Hsu, T., Soo, C., Zhang, B., Wang, J. Z., et al. (2007). Synergistic effects of Nell-1 and BMP-2 on the osteogenic differentiation of myoblasts. Journal of Bone and Mineral Research, 22, 918–930.
Altmann, F., Staudacher, E., Wilson, I. B., & Marz, L. (1999). Insect cells as hosts for the expression of recombinant glycoproteins. Glycoconjugate Journal, 16, 109–123.
Julenius, K., Molgaard, A., Gupta, R., & Brunak, S. (2005). Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites. Glycobiology, 15, 153–164.
Elbein, A. D. (1987). Inhibitors of the biosynthesis and processing of N-linked oligosaccharide chains. Annual Review of Biochemistry, 56, 497–534.
Kuan, S. F., Byrd, J. C., Basbaum, C., & Kim, Y. S. (1989). Inhibition of mucin glycosylation by aryl-N-acetyl-alpha-galactosaminides in human colon cancer cells. Journal of Biological Chemistry, 264, 19271–19277.
Fleury, D., Gillard, C., Lebhar, H., Vayssiere, B., Touitou, R., Rawadi, G., et al. (2008). Expression, purification and characterization of murine Dkk1 protein. Protein Expression and Purification, 60, 74–81.
Zhang, X., Carpenter, D., Bokui, N., Soo, C., Miao, S., Truong, T., et al. (2003). Overexpression of Nell-1, a craniosynostosis-associated gene, induces apoptosis in osteoblasts during craniofacial development. Journal of Bone and Mineral Research, 18, 2126–2134.
Timpl, R., Tisi, D., Talts, J. F., Andac, Z., Sasaki, T., & Hohenester, E. (2000). Structure and function of laminin LG modules. Matrix Biology, 19, 309–317.
Salasznyk, R. M., Williams, W. A., Boskey, A., Batorsky, A., & Plopper, G. E. (2004). Adhesion to vitronectin and collagen I promotes osteogenic differentiation of human mesenchymal stem cells. Journal of Biomedicine and Biotechnology, 2004, 24–34.
Klees, R. F., Salasznyk, R. M., Kingsley, K., Williams, W. A., Boskey, A., & Plopper, G. E. (2005). Laminin-5 induces osteogenic gene expression in human mesenchymal stem cells through an ERK-dependent pathway. Molecular Biology of the Cell, 16, 881–890.
Kundu, A. K., & Putnam, A. J. (2006). Vitronectin and collagen I differentially regulate osteogenesis in mesenchymal stem cells. Biochemical and Biophysical Research Communications, 347, 347–357.
Klees, R. F., Salasznyk, R. M., Ward, D. F., Crone, D. E., Williams, W. A., Harris, M. P., et al. (2008). Dissection of the osteogenic effects of laminin-332 utilizing specific LG domains: LG3 induces osteogenic differentiation, but not mineralization. Experimental Cell Research, 314, 763–773.
Tsai, K. S., Kao, S. Y., Wang, C. Y., Wang, Y. J., Wang, J. P., & Hung, S. C. (2010). Type I collagen promotes proliferation and osteogenesis of human mesenchymal stem cells via activation of ERK and Akt pathways. Journal of Biomedical Materials Research Part A, 94, 673–682.
Sottile, J., Selegue, J., & Mosher, D. F. (1991). Synthesis of truncated amino-terminal trimers of thrombospondin. Biochemistry, 30, 6556–6562.
Acknowledgments
This work was partially supported by a Grant-in-aid (nos. 18108003, 23510255) for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
About this article
Cite this article
Hasebe, A., Tashima, H., Ide, T. et al. Efficient Production and Characterization of Recombinant Human NELL1 Protein in Human Embryonic Kidney 293-F Cells. Mol Biotechnol 51, 58–66 (2012). https://doi.org/10.1007/s12033-011-9440-4
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12033-011-9440-4