Insertion of the designed helical linker led to increased expression of tf-based fusion proteins

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 ED(50) 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.

Fig. 1

High-level expression by the insertion of helical linker in both hGH-Tf and Tf-hGH fusion proteins as analyzed by Anti-hGH Western blot. Four fusion proteins with or without the inserted helical linker, expressed in serum free media, were analyzed by Western blot using goat anti-hGH monoclonal antibody (1:1000). The signal was detected using HRP-conjugated rabbit anti-goat secondary antibody (1:1000) and ECL reagents. The image was recorded and analyzed by ChemiDoc XBR (Bio-Rad). Lane 1: Tf (negative control); lane 2: hGH (10 ng); lane 3: hGH-Tf; lane 4: hGH-(H4)₂-Tf; lane 5: Tf-hGH; lane 6: Tf-(H4)₂-hGH.

Fig. 2

High-level expression by the insertion of helical linker in both hGH-Tf and Tf-hGH fusion proteins as analyzed by Anti-Tf Western blot. Four fusion proteins with or without the inserted helical linker, expressed in serum free media, were analyzed by Western blot using goat anti-Tf antibody (1:5000). The signal was detected using rabbit anti-goat secondary antibody conjugated to HRP (1:1000) and ECL reagents. The image was recorded and analyzed by ChemiDoc XBR. Lane 1: Tf (50 ng); lane 2: hGH (negative control); lane 3: hGH-Tf; lane 4: hGH-(H4)₂-Tf; lane 5: Tf-hGH; lane 6: Tf-(H4)₂-hGH.

Fig. 3

Helical linker insertion led to high-level expression in both hGH-Tf and Tf-hGH fusion protein as analyzed by SDS-PAGE. Same volume of conditioned media (5 μl) from the transfected HEK-293 cells were fractionated using SDS-PAGE, stained with Coomassie blue, and analyzed with ChemiDoc XBR. MM: molecular weight marker; lanes 1–3: Tf control; lane 4: hGH-Tf; lane 5: hGH-(H4)₂-Tf ; lane 6: Tf-hGH; lane 7: Tf-(H4)₂-hGH.

Fig. 4

Helical linker insertion led to high-level expression in both G-CSF-Tf and Tf-G-CSF fusion protein as analyzed by anti-G-CSF Western blot. 15 μl-conditioned media from the transfected HEK-293 cells were analyzed using anti-GCSF Western blot. Lane 1: G-CSF control; lane 2: pcDNA3.1(+) without the insert; lane 3: Tf-G-CSF; lane 4: Tf-(H4)-G-CSF; lane 5: Tf-(H4)₂-G-CSF; lane 6: Tf-r(H4)₂-G-CSF (fusion protein with a reversed (H4)₂ DNA sequence inserted, thus a non-helical linker in the product).