Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Paper
  • Published:

Production of Leukemia Inhibitory Factor in Escherichia coli by a Novel Procedure and Its Use in Maintaining Embryonic Stem Cells in Culture

Bio/Technology volume 7, pages 1157–1161 (1989)Cite this article

Abstract

Leukemia inhibitory factor [LIF] is a glycoprotein that is able to induce the differentiation of M1 myeloid leukemic cells and prevent the differentiation of murine embryonic stem [ES] cells. This report presents a simple method for the production and purification of large amounts of recombinant murine and human LIF in Escherichia coli. LIF is expressed initially as a fusion product with glutathione Stransferase, separated by a thrombin cleavage site. The fusion product can be rapidly purified on a glutathione-agarose affinity matrix and fully biologically active LIF released from the matrix by cleavage with thrombin. In this form LIF is suitable for many in vitro cell culture uses and in particular for ES cell culture. Further purification to homogeneity of thrombin-released LIF can be achieved by a single fractionation by reversed-phase high performance liquid chromatography; the purified product contains no detectable bacterial endotoxin. Evidence is presented that the intact fusion product has little or no biological activity but biologically active LIF is released by endogenous proteolytic cleavage.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Gearing, D.P., Gough, N.M., King, J.A., Hilton, D.J., Nicola, N.A., Simpson, R.J., Nice, E.C., Kelso, A., and Metcalf, D. 1987. Molecular cloning and expression of cDNA encoding a murine myeloid leukaemia inhibitory factor (LIF). EMBO J. 6:3995–4002.

    Article  CAS  Google Scholar 

  2. Gough, N.M., Gearing, D.P., King, J.A., Willson, T.A., Hilton, D.J., Nicola, N.A., and Metcalf, D. 1988. Molecular cloning and expression of the human gene homologous to murine myeloid leukaemia inhibitory factor (LIF). Proc. Natl. Acad. Sci. USA 85:2623–2627.

    Article  CAS  Google Scholar 

  3. Hilton, D.J., Nicola, N.A., and Metcalf, D. 1988. Purification of a murine leukemia inhibitory factor from Krebs Ascites cells. Anal. Biochem. 173:359–367.

    Article  CAS  Google Scholar 

  4. Hilton, D.J., Nicola, N.A. and Metcalf, D. 1988. Specific binding of murine leukemia inhibitory factor to normal and leukemic monocytic cells. Proc. Natl. Acad. Sci. USA 85:5971–5975.

    Article  CAS  Google Scholar 

  5. Metcalf, D., Hilton, D.J., and Nicola, N.A. 1988. Clonal analysis of the actions of the murine leukemia inhibitory factor on leukemic and normal murine hemopoietic cells. Leukemia 2:216–221.

    CAS  PubMed  Google Scholar 

  6. Gearing, D.P., King, J.A., and Gough, N.M. 1988. Complete sequence of murine myeloid leukaemia inhibitory factor. Nucl. Acids. Res. 16:9857.

    Article  CAS  Google Scholar 

  7. Simpson, R.J., Hilton, D.J., Nice, E.C., Rubira, M.R., Metcalf, D., Gearing, D.P., Gough, N.M. and Nicola, N.A. 1988. Structural characterization of a murine myeloid leukaemia inhibitory factor (LIF). Eur. J. Biochem. 175:541–547.

    Article  CAS  Google Scholar 

  8. Maekawa, T. and Metcalf, D. 1989. Clonal suppression of HL60 and U937 cells by recombinant human leukemia inhibitory factor in combination with GM-CSF or G-CSF. Leukemia 3:270–276.

    CAS  PubMed  Google Scholar 

  9. Tomida, M., Yamamoto-Yamaguchi, Y. and Hozumi, M. 1984. Purification of a factor inducing differentiation of mouse myeloid leukemic M1 cells from conditioned medium of mouse fibroblast L929 cells. J. Biol. Chem. 259:10978–10982.

    CAS  PubMed  Google Scholar 

  10. Williams, R.L., Hilton, D.J., Pease, S., Willson, T.A., Stewart, C.L., Gearing, D.P., Wagner, E.F., Metcalf, D., Nicola, N.A., and Gough, N.M. 1988. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336:684–687.

    Article  CAS  Google Scholar 

  11. Smith, A.G., Heath, J.K., Donaldson, D.D., Wong, G.G., Moreau, J., Stahl, M. and Rogers, D. 1988. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336:688–690.

    Article  CAS  Google Scholar 

  12. Thomas, K.R. and Capecchi, M.R. 1987. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51:503–512.

    Article  CAS  Google Scholar 

  13. Metcalf, D. and Gearing, D.P. 1989. A fatal syndrome in mice engrafted with cells producing high levels of the leukemia inhibitory factor (LIF). Proc. Natl. Acad. Sci. USA 86:5948–5952.

    Article  CAS  Google Scholar 

  14. Smith, D.B. and Johnson, K.S. 1988. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67:31–40.

    Article  CAS  Google Scholar 

  15. Smith, D.B., Davern, K.M., Board, P.G., Tiu, W.U., Garcia, E.G. and Mitchell, G.F. 1986. Mr 26000 antigen of Schistosoma japonicum recognized by resistant WEHI 129/J mice is a parasite glutathione S-transferase. Proc. Natl. Acad. Sci. USA 83:8703–8707.

    Article  CAS  Google Scholar 

  16. Smith, D.B., Davern, K.M., Board, P.G., Tiu, W.U., Garcia, E.G. and Mitchell, G.F. 1987. Mr 26000 antigen of Schistosoma japonicum recognized by resistant WEHI 129/J mice is a parasite glutathione S-transferase. Proc. Natl. Acad. Sci. USA 84:6541.

    Article  Google Scholar 

  17. Smith, D.B., Rubira, M.R., Simpson, R.J., Davern, K.M., Tiu, W.U., Board, P.G., and Mitchell, G.F. 1988. Expression of an enzymatically-active parasite molecule in Escherichia coli: Schistosoma japonicum glutathione S-transferase. Mol. Biochem. Parasitol. 27:249–256.

    Article  CAS  Google Scholar 

  18. Yanisch-Perron, C., Vieira, J. and Messing, J. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119.

    Article  CAS  Google Scholar 

  19. Maniatis, T., Fritsch, E.F. and Sambrook, J. 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

    Google Scholar 

  20. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685.

    Article  CAS  Google Scholar 

  21. Morrisey, J.H. 1981. Silver stain for proteins in polyacrylamide gels: A modified procedure with enhanced uniform sensitivity. Anal. Biochem. 117:307–310.

    Article  Google Scholar 

  22. Bernstine, E.G., Hooper, M.L., Grandchamp, S. and Ephrussi, B. 1973. Alkaline phosphatase activity in mouse teratoma. Proc. Natl. Acad. Sci. USA 70:3899–3903.

    Article  CAS  Google Scholar 

  23. Burstone, M.S. 1962. Enzyme Histochemistry and Its Application in the Study of Neoplasias, p. 88–113. Academic Press, New York.

  24. Doetschman, T.C., Eistetter, H., Katz, M., Schmidt, W. and Kemler, R. 1985. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp. Morphol. 87:27–45.

    CAS  Google Scholar 

  25. Smith, A.G., and Hooper, M.L. 1987. Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of murine embryonal carcinoma and embryonic stem cells. Dev. Biol. 121:1–9.

    Article  CAS  Google Scholar 

  26. Hopp, T.P., Prickett, K.S., Price, V.L., Libby, R.T., March, C.J., Cerretti, D.P., Urdal, D.L., and Conlon, P.J. 1988. A short polypeptide marker sequence useful for recombinant protein identification and purification. Bio Technology 6:1204–1210.

    Article  CAS  Google Scholar 

  27. Leutz, A., Damm, K., Sterneck, E., Kowenz, E., Ness, S., Frank, R., Gausepohl, H., Pan, Y-C.E., Smart, J., Hayman, M., Graf, T. 1989. Molecular cloning of the chicken myelomonocytic growth factor (cMGF) reveals relationship to interleukin 6 and granulocyte colony stimulating factor. EMBO J 8:175–181.

    Article  CAS  Google Scholar 

  28. Nagai, K. and Thogerson, H.C. 1987. Synthesis and sequence specific proteolysis of hybrid proteins produced by Escherichia coli. Methods Enzymol. 153:461–481.

    Article  CAS  Google Scholar 

  29. Levin, J., Tomasulo, P.A. and Oser, R.S. 1970. Detection of endotoxin human blood and demonstration of an inhibitor. J. Lab. Clin. Med. 75:903.

    CAS  PubMed  Google Scholar 

Download references

Author information

Author notes

  1. R. Lindsay Williams

    Present address: Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, 3050, Australia

  2. David P. Gearing: Corresponding author.

Authors and Affiliations

  1. The Walter and Eliza Hall Institute of Medical Research, Parkville, 3050, Australia

    David P. Gearing, Nicos A. Nicola, Donald Metcalf, Simon Foote, Tracy A. Willson & Nicholas M. Gough

  2. European Molecular Biology Laboratory, Meyerhofstrasse 1, 6900, Heidelberg, West Germany

    R. Lindsay Williams

Authors
  1. David P. Gearing
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nicos A. Nicola
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Donald Metcalf
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Simon Foote
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tracy A. Willson
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Nicholas M. Gough
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. R. Lindsay Williams
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gearing, D., Nicola, N., Metcalf, D. et al. Production of Leukemia Inhibitory Factor in Escherichia coli by a Novel Procedure and Its Use in Maintaining Embryonic Stem Cells in Culture. Nat Biotechnol 7, 1157–1161 (1989). https://doi.org/10.1038/nbt1189-1157

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1189-1157

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing