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Design of Recombinant Stem Cell Factor–macrophage Colony Stimulating Factor Fusion Proteins and their Biological Activity In Vitro

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Summary

Stem cell factor (SCF) and macrophage colony stimulating factor (M-CSF) can act in synergistic way to promote the growth of mononuclear phagocytes. SCF–M-CSF fusion proteins were designed on the computer using the Homology and Biopolymer modules of the software packages InsightII. Several existing crystal structures were used as templates to generate models of the complexes of receptor with fusion protein. The structure rationality of the fusion protein incorporated a series of flexible linker peptide was analyzed on InsightII system. Then, a suitable peptide GGGGSGGGGSGG was chosen for the fusion protein. Two recombinant SCF–M-CSF fusion proteins were generated by construction of a plasmid in which the coding regions of human SCF (1–165aa) and M-CSF (1–149aa) cDNA were connected by this linker peptide coding sequence followed by subsequent expression in insect cell. The results of Western blot and activity analysis showed that these two recombinant fusion proteins existed as a dimer with a molecular weight of ~84 KD under non-reducing conditions and a monomer of ~42 KD at reducing condition. The results of cell proliferation assays showed that each fusion protein induced a dose-dependent proliferative response. At equimolar concentration, SCF/M-CSF was about 20 times more potent than the standard monomeric SCF in stimulating TF-1 cell line growth, while M-CSF/SCF was 10 times of monomeric SCF. No activity difference of M-CSF/SCF or SCF/M-CSF to M-CSF (at same molar) was found in stimulating the HL-60 cell linear growth. The synergistic effect of SCF and M-CSF moieties in the fusion proteins was demonstrated by the result of clonogenic assay performed with human bone mononuclear, in which both SCF/M-CSF and M-CSF/SCF induced much higher number of CFU-M than equimolar amount of SCF or M-CSF or that of two cytokines mixture.

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References

  1. F. Herodin P. Bourin J.F. Mayol J.J. Lataillade M. Drouet (2003) Blood 101 2609 Occurrence Handle10.1182/blood-2002-06-1634 Occurrence Handle12468435

    Article  PubMed  Google Scholar 

  2. B.M. Curtis D.E. Williams H.E. Broxmeyer J. Dunn T. Farrah E. Jeffery W. Clevenger P. deRoos U. Martin D. Friend (1991) Proc. Natl. Acad. Sci. USA. 88 5809 Occurrence Handle1829529

    PubMed  Google Scholar 

  3. A. Coscarella C. Carloni R. Liddi S. Mauro S. Novelli A. Mele B. Masella M. Valtieri R. Santis ParticleDe (1997) Eur. J. Haematol. 59 238 Occurrence Handle9338622

    PubMed  Google Scholar 

  4. N.S. Weich J. Tullai E. Guido M. McMahon L.K. Jolliffe A.F. Lopez M.A. Vadas P.A. Lowry P.J. Quesenberry J. Rosen (1993) Exp. Hematol. 21 647 Occurrence Handle8513865

    PubMed  Google Scholar 

  5. M.R. Difalco L.F. Congote (1997) Biochem. J. 326 407 Occurrence Handle9291112

    PubMed  Google Scholar 

  6. F. Rock M. Everett M. Klein (1992) Protein Eng. 5 583 Occurrence Handle1438170

    PubMed  Google Scholar 

  7. C. Virginia Broudy (1997) Blood 90 1345 Occurrence Handle9269751

    PubMed  Google Scholar 

  8. J. Brandt R.A. Briddell E.F. Srour T.B. Leemhuis R. Hoffman (1992) Blood 79 634 Occurrence Handle1370637

    PubMed  Google Scholar 

  9. G. Andrews G.H. Knitter S.H. Bartelmex K.E. Langley D. Farrar R.W. Hendren F.R. Appelbaum I.D. Bernstein K.M. Zsebo (1991) Blood 78 2715 Occurrence Handle1824263

    PubMed  Google Scholar 

  10. I.D. Bernstein R.G. Andrews K.M. Zsebo (1991) Blood 77 2316 Occurrence Handle1710148

    PubMed  Google Scholar 

  11. A.R. Migliaccio M.L. Druzin P-J.V. Giardina K.M. Zsebo J.W. Adasmson (1992) Blood 79 2620 Occurrence Handle1375106

    PubMed  Google Scholar 

  12. D.N. Haylock L.B. To T.L. Dowse C.A. Juttner P.J. Simmons (1992) Blood 80 1405 Occurrence Handle1381625

    PubMed  Google Scholar 

  13. I.K. McNiece K.E. Langley K.M. Zsebo (1991) Exp. Hematol. 19 226 Occurrence Handle1704845

    PubMed  Google Scholar 

  14. S.J. Galli K.M. E. Zsebo Geissler (1994) Adv. Immunol. 55 96

    Google Scholar 

  15. I.K. McNiece R.A. Briddell (1995) J. Leukeo. Biol. 58 14

    Google Scholar 

  16. H.S. Lu C.L. Clogston J. Wypych P.R. Fausset S. Lauren E.A. Mendiaz K.M. Zsebo K.E. Langley (1991) J. Biol. Chem. 266 8102 Occurrence Handle1708771

    PubMed  Google Scholar 

  17. J.V. Matous K. Langley K. Kaushansky (1996) Blood 88 437 Occurrence Handle8695790

    PubMed  Google Scholar 

  18. K.E. Langley E.A. Mendiaz N. Liu L.O. Narhi L. Zeni C.M. Parseghian C.L. Clogston I. Leslie J.A. Pope H.S. Lu et al. (1994) Arch. Biochem. Biophys. 311 55 Occurrence Handle10.1006/abbi.1994.1208 Occurrence Handle7514387

    Article  PubMed  Google Scholar 

  19. Y.R. Hsu G.M. Wu E.A. Mendiaz R. Syed J. Wypych R. Toso M.B. Mann T.C. Boone L.O. Narhi H.S. Lu K.E Langley (1997) J. Biol. Chem. 272 6406 Occurrence Handle10.1074/jbc.272.10.6406 Occurrence Handle9045664

    Article  PubMed  Google Scholar 

  20. K.H. Nocka B.A. Levine J.L. Ko P.M. Burch B.E. Landgraf R. Segal R. Lobell (1997) Blood 90 3874 Occurrence Handle9354654

    PubMed  Google Scholar 

  21. F.J. Bot L. Eijk ParticleVan L. Broeders L.A. Aarden B. Löwenberg (1989) Blood 7 435

    Google Scholar 

  22. M.K. Warren P. Ralph (1986) J. Immunol. 137 2281 Occurrence Handle2428865

    PubMed  Google Scholar 

  23. A. Teranishi S. Akada S. Saito K. Hatake H. Morikawa (2002) Int. Immunopharmacol. 2 83 Occurrence Handle10.1016/S1567-5769(01)00149-7 Occurrence Handle11789672

    Article  PubMed  Google Scholar 

  24. T. Hidaka S. Akada A. Teranishi (2003) Cancer Sci. 94 814 Occurrence Handle10.1111/j.1349-7006.2003.tb01524.x Occurrence Handle12967481

    Article  PubMed  Google Scholar 

  25. M.J. Sweet D.A. Hume (2003) Arch. Immunol. Ther. Exp. (Warsz) 51 169

    Google Scholar 

  26. K. Mizutani S. Takeuchi Y. Ohashi (2003) Oncol. Rep. 10 127 Occurrence Handle12469157

    PubMed  Google Scholar 

  27. C.W. RRettenmier C.J. Sherr (1989) Hematol/Oncol Clin North Am 3 479

    Google Scholar 

  28. P. Fixe V. Praloran (1997) Eur. Cytokine Netw. 8 125 Occurrence Handle9262961

    PubMed  Google Scholar 

  29. D. Metcalf N.A. Nicola (1991) Proc. Natl. Acad. Sci. USA 88 6239 Occurrence Handle1712485

    PubMed  Google Scholar 

  30. M.H. Clare D.W. Anthony S. Nicholls K. Zsebo T.D. Michael (1992) Blood 80 2230 Occurrence Handle1384798

    PubMed  Google Scholar 

  31. E.N. Sian W. Sandra M.A. Rachel M. Jaleel D.W. Anthony (1999) Exp. Hematol. 27 663 Occurrence Handle10.1016/S0301-472X(98)00072-1 Occurrence Handle10210324

    Article  PubMed  Google Scholar 

  32. S. Lev J. Blechman S.-I. Nishikawa D. Givol Y. Yarden (1993) Mol.Cell Biol. 13 2224 Occurrence Handle7681144

    PubMed  Google Scholar 

  33. S. Lev J. Blechman S.-I. Nishikawa D. Givol Y. Yarden (1993) Mol. Cell Biol. 13 2224 Occurrence Handle7681144

    PubMed  Google Scholar 

  34. J. Yang C.Q. Liu (2000) Acta Pharmacologica Sinica 21 IssueID1 29–34 Occurrence Handle11263244

    PubMed  Google Scholar 

  35. J. Yang C.Y. Zhan X.C. Dong K. Yang F.X. Wang (2004) Acta Pharmacologica Sinica 25 IssueID8 1096–1104 Occurrence Handle15301746

    PubMed  Google Scholar 

  36. C. Wiesmann G. Fuh H.W. Christinger C. Eigenbrot J.A. Wells A.M. Vos Particlede (1997) Cell 28 695 Occurrence Handle10.1016/S0092-8674(00)80456-0

    Article  Google Scholar 

  37. Z. Zhang R. Zhang A. Joachimiak J. Schlessinger X.P. Kong (2000) Proc. Natl. Acad. Sci. USA 97 7732 Occurrence Handle10.1073/pnas.97.14.7732 Occurrence Handle10884405

    Article  PubMed  Google Scholar 

  38. X. Jiang O. Gurel E.A. Mendiaz G.W. Stearns C.L. Clogston H.S. Lu T.D. Osslund R.S. Syed K.E. Langley W.A. Hendrickson (2000) EMBO J. 19 3192 Occurrence Handle10.1093/emboj/19.13.3192 Occurrence Handle10880433

    Article  PubMed  Google Scholar 

  39. S.N. Savvides T. Boone K.P. Andrew (2000) Nat. Struct. Biol. 7 486 Occurrence Handle10.1038/75896 Occurrence Handle10881197

    Article  PubMed  Google Scholar 

  40. F. Balkwill (Eds) (2000) Cytokine Cell Biology EditionNumber3 Oxford University press Oxford

    Google Scholar 

  41. T. Mosmann (1983) J. Immunol. Meth. 65 55 Occurrence Handle10.1016/0022-1759(83)90303-4

    Article  Google Scholar 

  42. S.S. Tang G.G. Zheng K.F. Wu G.B. Chen H.Z. Liu Q. Rao (2001) Leuk. Res. 25 1107 Occurrence Handle10.1016/S0145-2126(01)00079-0 Occurrence Handle11684285

    Article  PubMed  Google Scholar 

  43. D. Metcalf (1970) J. Cell. Physiol. 76 89 Occurrence Handle10.1002/jcp.1040760113 Occurrence Handle5471044

    Article  PubMed  Google Scholar 

  44. A. Weaver D. Ryder D. Crowther T.M. Dexter N.G. Testa (1996) Blood 88 3323 Occurrence Handle8896396

    PubMed  Google Scholar 

  45. J.J. Costa G.D. Demetri T.J. Harrist A.M. Dvorak D.F. Hayes E.A. Merica D.M. Menchaca A.J. Gringeri L.B. Schwartz S.J. Galli (1996) J. Exp. Med. 183 2681 Occurrence Handle10.1084/jem.183.6.2681 Occurrence Handle8676090

    Article  PubMed  Google Scholar 

  46. K. Saigo T. Sugimoto T. Matsui R. Ryo S. Kumagai (2002) Clin. Lab. Haematol. 24 37 Occurrence Handle10.1046/j.1365-2257.2002.00156.x Occurrence Handle11843897

    Article  PubMed  Google Scholar 

  47. H. Mayani L.J. Guilbert S.C. Clark A. Janowska-Wieczorek (1991) Blood 78 651 Occurrence Handle1859881

    PubMed  Google Scholar 

  48. S.P. John W. Jie W. Jette G. Meredith E.A. Schwartz (1996) J. Biol. Chem. 271 6895 Occurrence Handle10.1074/jbc.271.49.31269 Occurrence Handle8636116

    Article  PubMed  Google Scholar 

  49. S.R. Leong H.B. Lowman J. Liu S. Shire L.E. Deforge B.L. Gillece-Castro R. McDowell C.A. Hebert (1997) Protein Sci. 6 609 Occurrence Handle9070443

    PubMed  Google Scholar 

  50. A. Landar B. Curry M.H. Parker R. DiGiacomo S.R. Indelicato T.L. Nagabhushan G. Rizzi M.R. Walter (2000) J. Mol. Biol. 299 169 Occurrence Handle10.1006/jmbi.2000.3734 Occurrence Handle10860730

    Article  PubMed  Google Scholar 

  51. H. Yueh-Rong Wu. Gay-May A. Elizabeth R.S. Mendiaz W.R. Jette B.M. Michael C. Thomas L.O. Boone S. Narhi Hsieng (1997) J. Biol. Chem. 272 6406 Occurrence Handle10.1074/jbc.272.10.6406 Occurrence Handle9045664

    Article  PubMed  Google Scholar 

  52. M.O. Glocker M. Kalkum R. Yamamoto J. Schreurs (1996) Biochemistry 35 14625 Occurrence Handle10.1021/bi961199o Occurrence Handle8931561

    Article  PubMed  Google Scholar 

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Authors and Affiliations

  1. School of life science and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 210093, Nanjing, People’s Republic of China

    Tao Chen, Jie Yang, Yuelang Wang, Chenyang Zhan, Yuhui Zang & Junchuan Qin

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  1. Tao Chen
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Correspondence to Jie Yang.

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Chen, T., Yang, J., Wang, Y. et al. Design of Recombinant Stem Cell Factor–macrophage Colony Stimulating Factor Fusion Proteins and their Biological Activity In Vitro. J Comput Aided Mol Des 19, 319–328 (2005). https://doi.org/10.1007/s10822-005-5686-x

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  • DOI: https://doi.org/10.1007/s10822-005-5686-x

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