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 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.