Production of recombinant CCN2 protein by mammalian cells

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Citation (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 languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages95-105
Number of pages11
Volume1489
DOIs
Publication statusPublished - 2017

Publication series

NameMethods in Molecular Biology
Volume1489
ISSN (Print)10643745

Fingerprint

Connective Tissue Growth Factor
Recombinant Proteins
Conditioned Culture Medium
HeLa Cells
Heparin
Electrophoresis
Chromatography
Western Blotting
Gels
Monoclonal Antibodies
Staining and Labeling
Escherichia coli
Proteins

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

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

Production of recombinant CCN2 protein by mammalian cells. / Nishida, Takashi; Kubota, Satoshi; Takigawa, Masaharu.

Methods in Molecular Biology. Vol. 1489 Humana Press Inc., 2017. p. 95-105 (Methods in Molecular Biology; Vol. 1489).

Research output: Chapter in Book/Report/Conference proceedingChapter

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