Production of recombinant CCN2 protein by mammalian cells

Takashi Nishida; Satoshi Kubota; Masaharu Takigawa

doi:10.1007/978-1-4939-6430-7_10

Production of recombinant CCN2 protein by mammalian cells

Takashi Nishida, Satoshi Kubota, Masaharu Takigawa

Research output: Chapter in Book/Report/Conference proceeding › Chapter

2 Citations (Scopus)

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.

Original language	English
Title of host publication	Methods in Molecular Biology
Publisher	Humana Press Inc.
Pages	95-105
Number of pages	11
DOIs	https://doi.org/10.1007/978-1-4939-6430-7_10
Publication status	Published - 2017

Publication series

Name	Methods in Molecular Biology
Volume	1489
ISSN (Print)	1064-3745

Keywords

ASF serum-free medium 104
Anti-CCN2 affinity chromatography
CCN2 expression vector
HeLa cells
Heparin affinity chromatography
Recombinant CCN2 protein (rCCN2)
Stable transfection

ASJC Scopus subject areas

Molecular Biology
Genetics

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Nishida, T., Kubota, S., & Takigawa, M. (2017). Production of recombinant CCN2 protein by mammalian cells. In Methods in Molecular Biology (pp. 95-105). (Methods in Molecular Biology; Vol. 1489). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-6430-7_10

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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.",

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