Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Sep 1;326(Pt 2):407–413. doi: 10.1042/bj3260407

Preparation of a recombinant chimaera of insulin-like growth factor II and interleukin 3 with high proliferative potency for haemopoietic cells.

M R Difalco 1, L F Congote 1
PMCID: PMC1218685  PMID: 9291112

Abstract

We have found that a slightly modified insulin-like growth factor II (IGF II) consisting of a chimaera of bombyxin and human IGF II (BOMIGF) is properly secreted in insect cells by using the baculovirus expression system. Human interleukin 3 (IL-3) was attached to the C-terminal amino acid residue of BOMIGF with peptide linkers containing five or twelve residues. Only the chimaera with the 12-residue linker had biological activities of both IGF II and IL-3. The chimaera had a significantly higher mitogenic activity than IL-3 in cell cultures of the human haemopoietic cell line TF-1 and its effect could be observed even at femtomolar concentrations. It was also able to stimulate thymidine incorporation in IGF II-dependent bovine fetal erythroid cells. The chimaera significantly increased the number of macroscopic haemopoietic colonies in cultures of human peripheral blood in comparison with IL-3 or mixtures of IL-3 and BOMIGF in vitro. Subcutaneous injection of a BOMIGF-mouse IL-3 chimaera in normal C57BL/6 mice resulted in a significant increase of the number of spleen stem cells producing macroscopic haemopoietic colonies. This new system for the biosynthesis of IGF-cytokine fusion proteins in insect cells might prove advantageous for the low-cost and high-yield production of molecules with complementary or synergistic biological activities.

Full Text

The Full Text of this article is available as a PDF (520.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barry S. C., Bagley C. J., Phillips J., Dottore M., Cambareri B., Moretti P., D'Andrea R., Goodall G. J., Shannon M. F., Vadas M. A. Two contiguous residues in human interleukin-3, Asp21 and Glu22, selectively interact with the alpha- and beta-chains of its receptor and participate in function. J Biol Chem. 1994 Mar 18;269(11):8488–8492. [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Burgaud J. L., Baserga R. Intracellular transactivation of the insulin-like growth factor I receptor by an epidermal growth factor receptor. Exp Cell Res. 1996 Mar 15;223(2):412–419. doi: 10.1006/excr.1996.0096. [DOI] [PubMed] [Google Scholar]
  4. Cohen M. C., Cohen S. Cytokine function: a study in biologic diversity. Am J Clin Pathol. 1996 May;105(5):589–598. doi: 10.1093/ajcp/105.5.589. [DOI] [PubMed] [Google Scholar]
  5. Congote L. F., Brox A., Lin F. K., Lu H. S., Fauser A. A. The N-terminal sequence of the major erythropoietic factor of an anephric patient is identical to insulin-like growth factor I. J Clin Endocrinol Metab. 1991 Mar;72(3):727–729. doi: 10.1210/jcem-72-3-727. [DOI] [PubMed] [Google Scholar]
  6. Congote L. F. Effects of insulin-like growth factor I, platelet-derived growth factor, fibroblast growth factor, and transforming growth factor-beta on thymidine incorporation into fetal liver cells. Exp Hematol. 1987 Oct;15(9):936–941. [PubMed] [Google Scholar]
  7. Congote L. F. Extraction from fetal bovine serum of erythrotropin, an erythroid cell-stimulating factor. Anal Biochem. 1984 Aug 1;140(2):428–433. doi: 10.1016/0003-2697(84)90189-1. [DOI] [PubMed] [Google Scholar]
  8. Congote L. F. Increased heparin binding by site directed mutagenesis of a recombinant chimera of bombyxin and insulin-like growth factor II. Biochim Biophys Acta. 1995 Apr 13;1243(3):538–542. doi: 10.1016/0304-4165(94)00217-l. [DOI] [PubMed] [Google Scholar]
  9. Congote L. F. Isolation of two biologically active peptides, erythrotropin I and erythrotropin II from fetal calf intestine. Biochem Biophys Res Commun. 1983 Sep 15;115(2):477–483. doi: 10.1016/s0006-291x(83)80169-7. [DOI] [PubMed] [Google Scholar]
  10. Congote L. F., Li Q. Accurate processing and secretion in the baculovirus expression system of an erythroid-cell-stimulating factor consisting of a chimaera of insulin-like growth factor II and an insect insulin-like peptide. Biochem J. 1994 Apr 1;299(Pt 1):101–107. doi: 10.1042/bj2990101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Congote L. F. Similarities in structure and function of bovine serum erythrotropin and human insulin-like growth factor II: two fetal erythroid cell stimulating factors. Biochem Biophys Res Commun. 1985 Jan 31;126(2):653–659. doi: 10.1016/0006-291x(85)90234-7. [DOI] [PubMed] [Google Scholar]
  12. Curtis B. M., Williams D. E., Broxmeyer H. E., Dunn J., Farrah T., Jeffery E., Clevenger W., deRoos P., Martin U., Friend D. Enhanced hematopoietic activity of a human granulocyte/macrophage colony-stimulating factor-interleukin 3 fusion protein. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5809–5813. doi: 10.1073/pnas.88.13.5809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dubé S., Fisher J. W., Powell J. S. Glycosylation at specific sites of erythropoietin is essential for biosynthesis, secretion, and biological function. J Biol Chem. 1988 Nov 25;263(33):17516–17521. [PubMed] [Google Scholar]
  14. Hober S., Forsberg G., Palm G., Hartmanis M., Nilsson B. Disulfide exchange folding of insulin-like growth factor I. Biochemistry. 1992 Feb 18;31(6):1749–1756. doi: 10.1021/bi00121a024. [DOI] [PubMed] [Google Scholar]
  15. Jones R. J., Wagner J. E., Celano P., Zicha M. S., Sharkis S. J. Separation of pluripotent haematopoietic stem cells from spleen colony-forming cells. Nature. 1990 Sep 13;347(6289):188–189. doi: 10.1038/347188a0. [DOI] [PubMed] [Google Scholar]
  16. Kurtz A., Jelkmann W., Bauer C. A new candidate for the regulation of erythropoiesis. Insulin-like growth factor I. FEBS Lett. 1982 Nov 22;149(1):105–108. doi: 10.1016/0014-5793(82)81081-8. [DOI] [PubMed] [Google Scholar]
  17. Narhi L. O., Hua Q. X., Arakawa T., Fox G. M., Tsai L., Rosenfeld R., Holst P., Miller J. A., Weiss M. A. Role of native disulfide bonds in the structure and activity of insulin-like growth factor 1: genetic models of protein-folding intermediates. Biochemistry. 1993 May 18;32(19):5214–5221. doi: 10.1021/bi00070a033. [DOI] [PubMed] [Google Scholar]
  18. Patchen M. L., Fischer R., MacVittie T. J., Seiler F. R., Williams D. E. Mast cell growth factor (C-kit ligand) in combination with granulocyte-macrophage colony-stimulating factor and interleukin-3: in vivo hemopoietic effects in irradiated mice compared to in vitro effects. Biotherapy. 1993;7(1):13–26. doi: 10.1007/BF01878150. [DOI] [PubMed] [Google Scholar]
  19. Rodriguez-Tarduchy G., Collins M. K., García I., López-Rivas A. Insulin-like growth factor-I inhibits apoptosis in IL-3-dependent hemopoietic cells. J Immunol. 1992 Jul 15;149(2):535–540. [PubMed] [Google Scholar]
  20. Rubin R., Baserga R. Insulin-like growth factor-I receptor. Its role in cell proliferation, apoptosis, and tumorigenicity. Lab Invest. 1995 Sep;73(3):311–331. [PubMed] [Google Scholar]
  21. Sakano K., Enjoh T., Numata F., Fujiwara H., Marumoto Y., Higashihashi N., Sato Y., Perdue J. F., Fujita-Yamaguchi Y. The design, expression, and characterization of human insulin-like growth factor II (IGF-II) mutants specific for either the IGF-II/cation-independent mannose 6-phosphate receptor or IGF-I receptor. J Biol Chem. 1991 Nov 5;266(31):20626–20635. [PubMed] [Google Scholar]
  22. Samuelsson E., Moks T., Nilsson B., Uhlen M. Enhanced in vitro refolding of insulin-like growth factor I using a solubilizing fusion partner. Biochemistry. 1994 Apr 12;33(14):4207–4211. doi: 10.1021/bi00180a013. [DOI] [PubMed] [Google Scholar]
  23. Saxena S. K., Crouse D. A., Sharp J. G. Effect of systemic interleukin-3 administration on epithelial cell proliferation in mouse intestine. Life Sci. 1993;53(6):473–477. doi: 10.1016/0024-3205(93)90698-3. [DOI] [PubMed] [Google Scholar]
  24. Schwartz G. N., Hudgins W. R., Perdue J. F. Glycosylated insulin-like growth factor II promoted expansion of granulocyte-macrophage colony-forming cells in serum-deprived liquid cultures of human peripheral blood cells. Exp Hematol. 1993 Oct;21(11):1447–1454. [PubMed] [Google Scholar]
  25. Shimon I., Shpilberg O. The insulin-like growth factor system in regulation of normal and malignant hematopoiesis. Leuk Res. 1995 Apr;19(4):233–240. doi: 10.1016/0145-2126(94)00133-u. [DOI] [PubMed] [Google Scholar]
  26. Tsarfaty G., Longo D. L., Murphy W. J. Human insulin-like growth factor I exerts hematopoietic growth-promoting effects after in vivo administration. Exp Hematol. 1994 Dec;22(13):1273–1277. [PubMed] [Google Scholar]
  27. Vadhan-Raj S., Broxmeyer H. E., Andreeff M., Bandres J. C., Buescher E. S., Benjamin R. S., Papadopoulos N. E., Burgess A., Patel S., Plager C. In vivo biologic effects of PIXY321, a synthetic hybrid protein of recombinant human granulocyte-macrophage colony-stimulating factor and interleukin-3 in cancer patients with normal hematopoiesis: a phase I study. Blood. 1995 Sep 15;86(6):2098–2105. [PubMed] [Google Scholar]
  28. Valentinis B., Baserga R. The IGF-I receptor protects tumor cells from apoptosis induced by high concentrations of serum. Biochem Biophys Res Commun. 1996 Jul 16;224(2):362–368. doi: 10.1006/bbrc.1996.1034. [DOI] [PubMed] [Google Scholar]
  29. Wang L. M., Michieli P., Lie W. R., Liu F., Lee C. C., Minty A., Sun X. J., Levine A., White M. F., Pierce J. H. The insulin receptor substrate-1-related 4PS substrate but not the interleukin-2R gamma chain is involved in interleukin-13-mediated signal transduction. Blood. 1995 Dec 1;86(11):4218–4227. [PubMed] [Google Scholar]
  30. Werner H., Adamo M., Roberts C. T., Jr, LeRoith D. Molecular and cellular aspects of insulin-like growth factor action. Vitam Horm. 1994;48:1–58. doi: 10.1016/s0083-6729(08)60495-1. [DOI] [PubMed] [Google Scholar]
  31. Williams D. E., Dunn J. T., Park L. S., Frieden E. A., Seiler F. R., Farese A. M., Macvittie T. J. A GM-CSF/IL-3 fusion protein promotes neutrophil and platelet recovery in sublethally irradiated rhesus monkeys. Biotechnol Ther. 1993;4(1-2):17–29. [PubMed] [Google Scholar]
  32. Wu D. D., Nayar R., Keating A. Synergistic effect of stem cell factor with interleukin-3 or granulocyte-macrophage colony-stimulating factor on the proliferation of murine primitive hematopoietic progenitors. Exp Hematol. 1994 Jun;22(6):495–500. [PubMed] [Google Scholar]
  33. Yoshinouchi M., Miura M., Gaozza E., Li S. W., Baserga R. Basic fibroblast growth factor stimulates DNA synthesis in cells overexpressing the insulin-like growth factor-I receptor. Mol Endocrinol. 1993 Sep;7(9):1161–1168. doi: 10.1210/mend.7.9.8247018. [DOI] [PubMed] [Google Scholar]
  34. Zauli G., Vitale M., Re M. C., Furlini G., Zamai L., Falcieri E., Gibellini D., Visani G., Davis B. R., Capitani S. In vitro exposure to human immunodeficiency virus type 1 induces apoptotic cell death of the factor-dependent TF-1 hematopoietic cell line. Blood. 1994 Jan 1;83(1):167–175. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES