title: Production of Recombinant CCN2 Protein by Mammalian Cells.
abstract: Recombinant CCN2 protein (rCCN2) is available from many companies; however, most of them are produced in E. coli. To investigate true functions of rCCN2, glycosylated protein with proper folding needs to be used. Therefore, we use rCCN2 produced by mammalian cells. Conditioned medium (CM) of HeLa cells stably transfected with a CCN2 expression vector are collected, and the recombinant CCN2 protein produced and secreted into the CM is purified by two-step chromatography, first with a heparin affinity column and then with an anti-CCN2 affinity column prepared with a monoclonal antibody against CCN2. The purified rCCN2 shows the bands of 36-38 kDa with sliver staining after gel electrophoresis, which can be confirmed by Western blotting. This chapter describes these methods in detail.
authors.affiliation: Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama, 700-8525, Japan. tnishida@md.okayama-u.ac.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Nishida
authors.affiliation: Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama, 700-8525, Japan.|Department of MembraneBiochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kubota
authors.affiliation: Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama, 700-8525, Japan. takigawa@md.okayama-u.ac.jp.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Takigawa
mesh_terms: D055511:CCN Intercellular Signaling Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods; D015870:Gene Expression; D023061:Gene Order; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins; D014162:Transfection / Q000379:methods
chemical_list: D055511:CCN Intercellular Signaling Proteins; D011993:Recombinant Fusion Proteins
keywords: ASF serum-free medium 104; Anti-CCN2 affinity chromatography; CCN2 expression vector; HeLa cells; Heparin affinity chromatography; Recombinant CCN2 protein (rCCN2); Stable transfection