Pharmaceutical Research

, 26:523 | Cite as

Insertion of the Designed Helical Linker Led to Increased Expression of Tf-Based Fusion Proteins

  • Nurmamet Amet
  • Hsin-Fang Lee
  • Wei-Chiang ShenEmail author
Research Paper



To demonstrate a high-level expression of transferrin (Tf)-based fusion proteins by inserting a helical linker between two protein domains.


Tf-based fusion proteins were designed to contain oligonucleotides encoding a helical linker inserted between the protein domains. Plasmid constructs were transfected into HEK293 cells and the secreted fusion proteins were purified from conditioned serum free media. Expression was assessed using both SDS-PAGE and Western Blot using anti-hGH, G-CSF, or Tf antibodies; protein bands were analyzed using Quantity One software. The function of fusion proteins consisting of human growth hormone (hGH) and Tf was evaluated in Nb2 cell proliferation assays.


The fusion proteins containing a helical linker, hGH-(H4)2-Tf and Tf-(H4)2-hGH, were expressed 1.7-and 2.4-fold higher, respectively, with a twofold lower ED50 than the hGH-Tf fusion protein without a helical linker. The Tf-(H4)2-G-CSF fusion protein exhibited a greater expression with an 11.2-fold increase compared with Tf-G-CSF fusion protein.


The helical linker introduced in Tf-fusion proteins resulted in a high-level of expression with improved in vitro bioactivity. This approach provides a simple method to increase poor expression of other fusion proteins.


domain switch fusion protein G-CSF helical linker hGH increased-expression transferrin 



2 copies of helical linker [A(EAAAK)4A]2


granulocyte colony stimulating factor


granulocyte colony stimulating factor-transferrin recombinant fusion protein


human growth hormone


hGH-Tf fusion protein with two copies of helical linker


human growth hormone-transferrin recombinant fusion protein


1 copy of helical linker A(EAAAK)4A


human serum transferrin


transferrin-granulocyte colony stimulating factor recombinant fusion protein with Tf at N-terminus


transferrin-human growth hormone fusion protein with Tf at N-terminus


Tf-G-CSF fusion protein with a reversed (H4)2 DNA sequence inserted. Nb2 cells, rat T lymphoma cells that proliferate upon stimulation by the bioactive hGH


Tf-G-CSF fusion protein with 2 copies of helical linker


Tf-hGH fusion protein with 2 copies of helical linker


  1. 1.
    L. Baldi, D. L. Hacker, M. Adam, and F. M. Wurm. Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives. Biotechnol. Lett. 29:677–684 (2007). doi: 10.1007/s10529-006-9297-y.PubMedCrossRefGoogle Scholar
  2. 2.
    F. Wurm, and A. Bernard. Large-scale transient expression in mammalian cells for recombinant protein production. Curr. Opin. Biotechnol. 10:156–159 (1999). doi: 10.1016/S0958-1669(99)80027-5.PubMedCrossRefGoogle Scholar
  3. 3.
    F. M. Wurm. Production of recombinant protein therapeutics in cultivated mammalian cells. Nat. Biotechnol. 22:1393–1398 (2004). doi: 10.1038/nbt1026.PubMedCrossRefGoogle Scholar
  4. 4.
    G. M. Subramanian, M. Fiscella, A. Lamousé-Smith, S. Zeuzem, and J. G. McHutchison. Albinterferon alpha-2b: a genetic fusion protein for the treatment of chronic hepatitis C. Nat. Biotechnol. 25:1411–1419 (2007). doi: 10.1038/nbt1364.PubMedCrossRefGoogle Scholar
  5. 5.
    C. Wu, H. Ying, C. Grinnell, S. Bryant, R. Miller, A. Clabbers, S. Bose, D. McCarthy, R. R. Zhu, and L. Santora. Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin. Nat. Biotechnol. 25:1290–1297 (2007). doi: 10.1038/nbt1345.PubMedCrossRefGoogle Scholar
  6. 6.
    B. Leader, Q. J. Baca, and D. E. Golan. Opinion: protein therapeutics: a summary and pharmacological classification. Nature Reviews Drug Discovery. 7:21–39 (2008). doi: 10.1038/nrd2399.PubMedCrossRefGoogle Scholar
  7. 7.
    M. Kavoosi, A. L. Creagh, D. G. Kilburn, and C. A. Haynes. Strategy for selecting and characterizing linker peptides for CBM9-tagged fusion proteins expressed in Escherichia coli. Biotechnol. Bioeng. 98:599–610 (2007). doi: 10.1002/bit.21396.PubMedCrossRefGoogle Scholar
  8. 8.
    K. D. Pryor, and B. Leiting. High-level expression of soluble protein in Escherichia coli using a His6-Tag and maltose-binding-protein double-affinity fusion system. Protein Expr. Purif. 10:309–319 (1997). doi: 10.1006/prep.1997.0759.PubMedCrossRefGoogle Scholar
  9. 9.
    Y. Maeda, H. Ueda, J. Kazami, G. Kawano, E. Suzuki, and T. Nagamune. Engineering of functional chimeric protein G-vargula luciferase. Anal. Biochem. 249:147–152 (1997). doi: 10.1006/abio.1997.2181.PubMedCrossRefGoogle Scholar
  10. 10.
    R. Arai, H. Ueda, A. Kitayama, N. Kamiya, and T. Nagamune. Design of the linkers which effectively separate domains of a bifunctional fusion protein. Protein Eng. 14:529–532 (2001). doi: 10.1093/protein/14.8.529.PubMedCrossRefGoogle Scholar
  11. 11.
    R. Arai, W. Wriggers, Y. Nishikawa, T. Nagamune, and T. Fujisawa. Conformations of variably linked chimeric proteins evaluated by synchrotron X-ray small-angle scattering. Proteins Structure Function and Bioinformatics. 57:829–838 (2004). doi: 10.1002/prot.20244.CrossRefGoogle Scholar
  12. 12.
    E. Park, R. M. Starzyk, J. P. McGrath, T. Lee, J. George, A. J. Schutz, P. Lynch, and S. D. Putney. Production and characterization of fusion proteins containing transferrin and nerve growth factor. J. Drug Target. 6:53–64 (1998).PubMedCrossRefGoogle Scholar
  13. 13.
    Y. Bai, and W. C. Shen. Improving the oral efficacy of recombinant granulocyte colony-stimulating factor and transferrin fusion protein by spacer optimization. Pharm. Res. 23:2116–2121 (2006). doi: 10.1007/s11095-006-9059-5.PubMedCrossRefGoogle Scholar
  14. 14.
    Y. Bai, D. K. Ann, and W.-C. Shen. Recombinant granulocyte colony-stimulating factor-transferrin fusion protein as an oral myelopoietic agent. Proc. Natl. Acad. Sci. 102:7292–7296 (2005). doi: 10.1073/pnas.0500062102.PubMedCrossRefGoogle Scholar
  15. 15.
    M. Ishikawa, A. Nimura, R. Horikawa, N. Katsumata, O. Arisaka, M. Wada, M. Honjo, and T. Tanaka. A novel specific bioassay for serum human growth hormone. J. Clin. Endocrinol. Metab. 85:4274–4279 (2000).PubMedCrossRefGoogle Scholar
  16. 16.
    C. R. Robinson, and R. T. Sauer. Optimizing the stability of single-chain proteins by linker length and composition mutagenesis. Proc. Natl. Acad. Sci. 95:5929–5934 (1998). doi: 10.1073/pnas.95.11.5929.PubMedCrossRefGoogle Scholar
  17. 17.
    S. Marqusee, and R. L. Baldwin. Helix stabilization by Glu-\cdots Lys+ salt bridges in short peptides of de novo design. Proc. Natl. Acad. Sci. U. S. A. 84:8898–8902 (1987). doi: 10.1073/pnas.84.24.8898.PubMedCrossRefGoogle Scholar
  18. 18.
    P. L. Pham, S. Perret, B. Cass, E. Carpentier, G. St-Laurent, L. Bisson, A. Kamen, and Y. Durocher. Transient gene expression in HEK293 cells: peptone addition posttransfection improves recombinant protein synthesis. Biotechnol. Bioeng. 90:332–344 (2005). doi: 10.1002/bit.20428.PubMedCrossRefGoogle Scholar
  19. 19.
    C. K. Crowell, Q. Qin, G. E. Grampp, R. A. Radcliffe, G. N. Rogers, and R. I. Scheinman. Sodium butyrate alters erythropoietin glycosylation via multiple mechanisms. Biotechnol. Bioeng. 99:201–213 (2008). doi: 10.1002/bit.21539.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  1. 1.Department of Pharmacology and Pharmaceutical SciencesUniversity of Southern California School of PharmacyLos AngelesUSA

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