Skip to main content
SpringerLink
Log in

Production of Recombinant CCN2 Protein by Mammalian Cells

  • Protocol
  • First Online:
CCN Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1489))

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Bork P (1993) The molecular architecture of a new family of growth regulators related to connective tissue growth factor. FEBS Lett 327:125–130

    Article  CAS  PubMed  Google Scholar 

  2. Brigstock DR (1999) The connective tissue growth factor/cystein-rich 61/nephroblastoma overexpressed (CCN) family. Endocr Rev 20:189–206

    CAS  PubMed  Google Scholar 

  3. Pennica D, Swanson TA, Welsh JW, Roy MA, Lawrence DA, Lee J, Brush J, Taneyhill LA, Deuel B, Lew M, Watanabe C, Cohen RL, Melhem MF, Finley GG, Quirke P, Goddard AD, Hillan KJ, Gurney AL, Botstein D, Levine AJ (1998) WISP genes are members of the connective tissue growth factor family that are up-regulated in wnt-1-transformed cells and aberrantly expressed in human colon tumors. Proc Natl Acad Sci U S A 95:14717–14722

    Google Scholar 

  4. Brigstock DR, Goldschmeding R, Katsube KI, Lam SC, Lau LF, Lyons KM, Naus C, Perbal B, Riser B, Takigawa M, Yeger H (2003) Proposal for a unified CCN nomenclature. Mol Pathol 56:127–128

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Nakanishi T, Nishida T, Shimo T, Kobayashi K, Kubo T, Tamatani T, Tezuka K, Takigawa M (2000) Effects of CTGF/Hcs24, a product of a hypertrophic chondrocyte-specific gene, on the proliferation and differentiation of chondrocytes in culture. Endocrinology 141:264–273

    CAS  PubMed  Google Scholar 

  6. Ivkovic S, Yoon BS, Popoff SN, Safadi FF, Libuda DE, Stephenson RC, Daluiski A, Lyons KM (2003) Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development 130:2779–2791

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Nishida T, Nakanishi T, Asano M, Shimo T, Takigawa M (2000) Effects of CTGF/Hcs24, a hypertrophic chondrocyte-specific gene product, on the proliferation and differentiation of osteoblastic cells in vitro. J Cell Physiol 184:197–206

    Article  CAS  PubMed  Google Scholar 

  8. Shimo T, Nakanishi T, Nishida T, Asano M, Kanyama M, Kuboki T, Tamatani T, Tezuka K, Takemura M, Matsumura T, Takigawa M (1999) Connective tissue growth factor induces the proliferation, migration, and tube formation of vascular endothelial cells in vitro, and angiogenesis in vivo. J Biochem 126:137–145

    Article  CAS  PubMed  Google Scholar 

  9. Nakata E, Nakanishi T, Kawai A, Asaumi K, Yamaai T, Asano M, Nishida T, Mitani S, Inoue H, Takigawa M (2002) Expression of connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) during fracture healing. Bone 31:441–447

    Google Scholar 

  10. Igarashi A, Nashiro K, Kikuchi K, Sato S, Ihn H, Fujimoto M, Grotendorst GR, Takehara K (1996) Connective tissue growth factor gene expression in tissue sections from localized scleroderma, keloid, and other fibrotic skin disorders. J Invest Dermatol 106:729–733

    Google Scholar 

  11. Takigawa M, Nakanishi T, Kubota S, Nishida T (2003) Role of CTGF/Hcs24/ecogenin in skeletal growth control. J Cell Physiol 194:256–266

    Article  CAS  PubMed  Google Scholar 

  12. Tamatani T, Kobayashi H, Tezuka K, Sakamoto S, Suzuki K, Nakanishi T, Takigawa M, Miyano T (1998) Establishment of the enzyme-linked immunosorbent assay for connective tissue growth factor (CTGF) and its detection in the sera of billiary atresia. Biochem Biophys Res Commun 251:748–752

    Google Scholar 

  13. Bradham DM, Igarashi A, Potter RL, Grotendorst GR (1991) Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10. J Cell Biol 114:1285–1294

    Google Scholar 

  14. Nishida T, Nakanishi T, Shimo T, Asano M, Hattori T, Tamatani T, Tezuka K, Takigawa M (1998) Demonstration of receptors specific for connective tissue growth factor on a human chondrocytic cell line (HCS-2/8). Biochem Biophys Res Commun 247:905–909

    Google Scholar 

  15. Nishida T, Kubota S, Nakanishi T, Kuboki T, Yosimichi G, Kondo S, Takigawa M (2002) CTGF/Hcs24, a hypertrophic chondrocyte-specific gene product, stimulates proliferation and differentiation, but not hypertrophy of cultured articular chondrocytes. J Cell Physiol 192:55–63

    Google Scholar 

  16. Nishida T, Kubota S, Fukunaga T, Kondo S, Yosimichi G, Nakanishi T, Takano-Yamamoto T, Takigawa M (2003) CTGF/Hcs24, hypertrophic chondrocyte-specific gene product, interacts with perlecan in regulating the proliferation and differentiation of chondrocytes. J Cell Physiol 196:265–275

    Article  CAS  PubMed  Google Scholar 

  17. Kawaki H, Kubota S, Suzuki A, Yamada T, Matsumura T, Mandai T, Yao M, Maeda T, Lyons KM, Takigawa M (2008) Functional requirement of CCN2 for intramembranous bone formation in embryonic mice. Biochem Biophys Res Commun 366:450–456

    Article  CAS  PubMed  Google Scholar 

  18. Nishida T, Emura K, Kubota S, Lyons KM, Takigawa M (2011) CCN family 2/connective tissue growth factor (CCN2/CTGF) promotes osteoclastogenesis via induction of and interaction with dendritic cell-specific transmembrane protein (DC-STAMP). J Bone Miner Res 26:351–363

    Article  CAS  PubMed  Google Scholar 

  19. Aoyama E, Kubota S, Khattab HM, Nishida T, Takigawa M (2015) CCN2 enhances RANKL-induced osteoclast differentiation via direct binding to RANK and OPG. Bone 73:242–248

    Article  CAS  PubMed  Google Scholar 

  20. Nishida T, Kondo S, Maeda A, Kubota S, Lyons KM, Takigawa M (2009) CCN family 2/connective tissue growth factor (CCN2/CTGF) regulates the expression of Vegf through Hif-1α expression in a chondrocytic cell line, HCS-2/8, under hypoxic condition. Bone 44:24–31

    Article  CAS  PubMed  Google Scholar 

  21. Nishida T, Kubota S, Aoyama E, Janune D, Lyons KM, Takigawa M (2015) CCN family protein 2 (CCN2) promotes the early differentiation, but inhibits the terminal differentiation of skeletal myoblasts. J Biochem 157:91–100

    Google Scholar 

Download references

Acknowledgments

This work was supported in part by grants from the programs Grants-in-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.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama, 700-8525, Japan

    Takashi Nishida D.D.S., Ph.D.

  2. Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama, 700-8525, Japan

    Satoshi Kubota & Masaharu Takigawa D.D.S., Ph.D.

  3. Department of MembraneBiochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan

    Satoshi Kubota

Authors
  1. Takashi Nishida D.D.S., Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Satoshi Kubota
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Masaharu Takigawa D.D.S., Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Takashi Nishida D.D.S., Ph.D. or Masaharu Takigawa D.D.S., Ph.D. .

Editor information

Editors and Affiliations

  1. Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama, Japan

    Masaharu Takigawa

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media New York

About this protocol

Cite this protocol

Nishida, T., Kubota, S., Takigawa, M. (2017). Production of Recombinant CCN2 Protein by Mammalian Cells. In: Takigawa, M. (eds) CCN Proteins. Methods in Molecular Biology, vol 1489. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6430-7_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6430-7_10

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6428-4

  • Online ISBN: 978-1-4939-6430-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation