RESEARCH Efficient Production and Characterization of Recombinant Human NELL1 Protein in Human Embryonic Kidney 293-F Cells Ai Hasebe • Hiroki Tashima • Teruhiko Ide • Masumi Iijima • Nobuo Yoshimoto • Kang Ting • Shun’ichi Kuroda • Tomoaki Niimi Published online: 5 August 2011 Ó Springer Science+Business Media, LLC 2011 Abstract NELL1 is a secretory protein that induces osteogenic differentiation and bone formation by osteo- blastic 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 path- ways. 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 hexa- histidine-tag were obtained by G418 selection of trans- fected 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 com- ponent. 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. Keywords NELL-1 Á Osteoblast differentiation Á Bone regeneration Á Extracellular matrix Introduction NELL1 [Nel (a gene strongly expressed in neural tissues encoding epidermal growth factor-like domain)-like mol- ecule 1] is a secretory protein that shows potent osteoin- ductive activity both in vivo and in vitro [1]. NELL1 was first isolated from human unilateral coronal craniosynos- tosis (CS) patients, in whom it is specifically upregulated within prematurely fusing sutures [2]. Transgenic mice overexpressing the NELL1 gene under the control of a cytomegalovirus (CMV) promoter exhibit a phenotype similar to that of human CS, while N-ethyl-N-nitrosourea- induced NELL1-deficient mice show cranial and vertebral skeletal defects, suggesting a role for NELL1 in bone and skeletal development [3, 4]. In committed osteoblasts or mesenchymal stem cells, NELL1 upregulation accelerates osteogenic differentiation [3, 5]. We recently showed that NELL1 transduces osteogenic signals through the Ras- mitogen-activated protein kinase (MAPK) pathway [6]. However, no candidate receptors for NELL1 have been identified. The human NELL1 gene encodes a polypeptide of 810 amino acids, which contains several structural motifs, including a laminin G (LG) domain [overlapping with an A. Hasebe Á H. Tashima Á T. Ide Á M. Iijima Á N. Yoshimoto Á S. Kuroda (&) Á T. Niimi (&) Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan e-mail: skuroda@agr.nagoya-u.ac.jp T. Niimi e-mail: tniimi@agr.nagoya-u.ac.jp K. Ting Dental and Craniofacial Research Institute, University of California Los Angeles, Los Angeles, CA 90095, USA 123 Mol Biotechnol (2012) 51:58–66 DOI 10.1007/s12033-011-9440-4