Production of recombinant CCN2 protein by mammalian cells

Research output: Chapter in Book/Report/Conference proceedingChapter

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

Publication series

NameMethods in Molecular Biology
Volume1489
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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  • Cite this

    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