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 |
| Volume | 1489 |
| DOIs | |
| Publication status | Published - 2017 |
Publication series
| Name | Methods in Molecular Biology |
|---|---|
| Volume | 1489 |
| ISSN (Print) | 10643745 |
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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
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 proceeding › Chapter
}
TY - CHAP
T1 - Production of recombinant CCN2 protein by mammalian cells
AU - Nishida, Takashi
AU - Kubota, Satoshi
AU - Takigawa, Masaharu
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 - Anti-CCN2 affinity chromatography
KW - ASF serum-free medium 104
KW - CCN2 expression vector
KW - HeLa cells
KW - Heparin affinity chromatography
KW - Recombinant CCN2 protein (rCCN2)
KW - Stable transfection
UR - http://www.scopus.com/inward/record.url?scp=84991628305&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84991628305&partnerID=8YFLogxK
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
VL - 1489
T3 - Methods in Molecular Biology
SP - 95
EP - 105
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -