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