TY - CHAP
T1 - Production of recombinant CCN2 protein by mammalian cells
AU - Nishida, Takashi
AU - Kubota, Satoshi
AU - Takigawa, Masaharu
N1 - Funding Information:
This work was supported in part by grants from the programs Grantsin-Aid for Scientific Research (C) to TN (#JP26462810) and to SK (#JP25462886) and Scientific Research (B) to MT (#JP15H05014) from the Japan Society for the Promotion of Sciences, Japan. We thank Drs. Takuya Tamatani and Katsunari Tezuka (Pharmaceutical Frontier Research Laboratories, Japan Tabacco, Inc., Yokohama, Japan) for helpful suggestions and technical assistance, as well as Miss Yoshiko Miyake for secretarial assistance.
PY - 2017
Y1 - 2017
N2 - 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.
AB - 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.
KW - ASF serum-free medium 104
KW - Anti-CCN2 affinity chromatography
KW - CCN2 expression vector
KW - HeLa cells
KW - Heparin affinity chromatography
KW - Recombinant CCN2 protein (rCCN2)
KW - Stable transfection
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U2 - 10.1007/978-1-4939-6430-7_10
DO - 10.1007/978-1-4939-6430-7_10
M3 - Chapter
C2 - 27734369
AN - SCOPUS:84991628305
T3 - Methods in Molecular Biology
SP - 95
EP - 105
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -