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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

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.

KEY WORDS

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

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

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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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