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

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Abstract

Purpose

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

Methods

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.

Results

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.

Conclusions

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.

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Abbreviations

(H4)2 :

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

G-CSF:

granulocyte colony stimulating factor

G-CSF-Tf:

granulocyte colony stimulating factor-transferrin recombinant fusion protein

hGH:

human growth hormone

hGH-(H4)2-Tf:

hGH-Tf fusion protein with two copies of helical linker

hGH-Tf:

human growth hormone-transferrin recombinant fusion protein

H4:

1 copy of helical linker A(EAAAK)4A

Tf:

human serum transferrin

Tf-G-CSF:

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

Tf-hGH:

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

Tf-r(H4)2-G-CSF:

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-(H4)2-G-CSF:

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

Tf-(H4)2-hGH:

Tf-hGH fusion protein with 2 copies of helical linker

References

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  3. F. M. Wurm. Production of recombinant protein therapeutics in cultivated mammalian cells. Nat. Biotechnol. 22:1393–1398 (2004). doi:10.1038/nbt1026.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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).

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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).

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Pharmacology and Pharmaceutical Sciences, University of Southern California School of Pharmacy, PSC 404B, 1985 Zonal Avenue, Los Angeles, California, 90089-9121, USA

    Nurmamet Amet, Hsin-Fang Lee & Wei-Chiang Shen

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  1. Nurmamet Amet
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  2. Hsin-Fang Lee
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  3. Wei-Chiang Shen
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Correspondence to Wei-Chiang Shen.

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Amet, N., Lee, HF. & Shen, WC. Insertion of the Designed Helical Linker Led to Increased Expression of Tf-Based Fusion Proteins. Pharm Res 26, 523–528 (2009). https://doi.org/10.1007/s11095-008-9767-0

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  • DOI: https://doi.org/10.1007/s11095-008-9767-0

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